Effects of Pistachio Consumption on Body Composition

Home , Pistachio

EFFECTS OF PISTACHIO CONSUMPTION ON BODY COMPOSITION,

SERUM LIPIDS, AND FATTY ACID INCORPORATION

INTO ERYTHROCYTE MEMBRANES

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Agriculture

Amy H. Towne

2014

SIGNATURE PAGE

THESIS: EFFECTS OF PISTACHIO CONSUMPTION ON BODY COMPOSITION, SERUM LIPIDS, AND FATTY ACID INCORPORATION INTO ERYTHROCYTE MEMBRANES

AUTHOR: Amy H. Towne

DATE SUBMITTED: Spring 2014

College of Agriculture

Dr. Bonny Burns-Whitmore ______Thesis Committee Chair Human Nutrition and Food Science

Dr. David Edens ______Human Nutrition and Food Science

Golandam Khayef, MS, RD ______Human Nutrition and Food Science

ACKNOWLEDGEMENTS

I would like to thank my advisor and thesis committee chair Dr. Bonny Burns-

Whitmore for allowing me the opportunity to not only partake in this research but also helping me learn and grow in the process. I would like to thank Golandam Khayef for not hesitating to join this committee and for her constructive and appreciated feedback. I would like to thank Dr. David Edens for joining my committee and immediately contributing his knowledge and resources to the success of this thesis. I would like to thank Dr. Spalding for his positivity and valuable contributions to this thesis as well.

Additionally, I would like to thank all of my professors during my graduate studies; each and every class has contributed to this thesis. Finally, I would like to thank my family for their love and support, and Jason Porter for his constant support throughout this process.

My gratitude goes to the volunteer subjects as well as research assistants, without you this research would not be possible.

iii

ABSTRACT

Risk of cardiovascular disease is lower in those with healthy serum lipid levels and healthy body composition. A diet high in nuts has been shown to improve or maintain body composition and serum lipid levels. The present study sought to determine the effects of regular pistachio nut consumption in young, healthy, free-living, female participants. This study utilized a cross-over design with two 10-week treatment periods, pistachio enriched diet (PE) in which 20% of daily calories were provided by pistachios and no-pistachio control diet (NP), separated by a 15-week washout period. At the beginning and end of each treatment period, following a 12-hour overnight fast, participants’ blood lipids, waist circumference, hip circumference, body weight and body composition using bioelectrical impedance analysis measurements were taken. There were no significant differences in body composition or serum lipid between PE and NP.

Baseline and post-treatment serum lipid and body composition values were within ideal ranges for lowered cardiovascular disease risk. Improvement was seen in the mean lowering of total cholesterol, LDL cholesterol and in total cholesterol to HDL cholesterol and in LDL cholesterol to HDL cholesterol (ratios), following the PE treatment. No significant change was seen between treatment groups in the incorporation of fatty acids into the erythrocyte membrane. There was a non-significant mean decrease in erythrocyte percent saturated fatty acid, MUFA, and n3 fatty acid following both treatments. A diet rich in pistachio nuts does not contribute to changes in body composition and may potentially improve serum lipids in young, free-living, healthy, females.

TABLE OF CONTENTS

Signature Page ...... ii

Acknowledgements ...... iii

Abstract ...... iv

List of Tables ...... vii

List of Figures ...... ix

Chapter 1: Introduction ...... 1

Statement of the Problem ...... 1

Definitions...... 7

Chapter 2: Literature Review ...... 10

Current Dietary Recommendations to Lower the Risk of CVD ...... 10

Chapter 3: Methodology ...... 31

Participants ...... 31

Data Collection ...... 38

Chapter 4: Research Findings ...... 46

Participant Characteristics ...... 46

Chapter 5: Discussion ...... 55

Research Basis ...... 55

Chapter 6: Conclusion...... 76

References ...... 79

Appendix A: IRB Approval Letter ...... 90

Appendix B: Sample Recruitment E-mail...... 91

Appendix C: Informed Consent Form...... 92

Appendix D: Initial Screening Questionnaire...... 99

Appendix E: International Physical Activity Questionnaire...... 102

Appendix F: Sample 24-hour Diet Record...... 105

Appendix G: Sample Unusual Diet Diary...... 108

Appendix H: Journal Article ...... 111

LIST OF TABLES

Table 1 Composition of the ATP III TLC Diet ...... 11

Table 2 Composition of the DASH Diet ...... 13

Table 3 Guideline from the AHA – Specific Dietary Intake Recommendations for Women ...... 14

Table 4 Dietary Guidelines for Americans – Macronutrient Recommendations - Females 19 – 50 ...... 16

Table 5 Details of Studies Examining the Effects of Regular Nut Consumption (Epidemiological)(n=8,865)...... 23

Table 6 Details of Studies Examining the Effects of Regular Nut Consumption (Epidemiological)(n=13,292)...... 24

Table 7 Details of Studies Examining the Effects of Regular Pistachio Consumption (Clinical)(n=10) ...... 25

Table 8 Details of Studies Examining the Effects of Regular Pistachio Consumption (Clinical)(n=44) ...... 26

Table 9 Details of Studies Examining the Effects of Regular Pistachio Consumption (Clinical)(n=15) ...... 27

Table 10 Details of Studies Examining the Effects of Regular Pistachio Consumption (Clinical)(n=28) ...... 28

Table 11 Details of Studies Examining the Effects of Regular Pistachio Consumption (Clinical)(n=60) ...... 29

Table 12 Dietary Guidelines for Americans – Macronutrient Recommendations - Females 19 – 50 ...... 30

Table 13 Mean Participant Baseline, Pre-pistachio Treatment, Anthropometric Characteristics ...... 48

Table 14 Mean Participant Baseline, Pre-pistachio Treatment, Serum Lipid Profile ...... 49

vii

Table 15 Mean Participant Baseline, Pre-pistachio Treatment, Erythrocyte Fatty Acid Percentages ...... 50

Table 16 Pre and Post PE Intervention Serum Lipid Means and AHA Advocated Serum Lipid Values for Women ...... 57

Table 17 Pre and Post NP Intervention Serum Lipid Means and AHA Advocated Serum Lipid Values for Women ...... 58

Table 18 Pre and Post PE Intervention Percent Fatty Acid of Erythrocyte Membrane ...... 73

Table 19 Pre and Post NP Intervention Percent Fatty Acid of Erythrocyte Membrane ...... 79

viii

LIST OF FIGURES

Figure 1 Summary of the crossover design employed in this study ...... 35

Figure 2 Summary of screening and enrollment demographics ...... 47

Figure 3 Pre and post 10-week pistachio enriched (PE) and no pistachio control (NP) diets ...... 61

Figure 4 Percent body fat change pre to post pistachio enriched diet (PE) and pre to post no-pistachio control diet (NP) ...... 62

Figure 5 Fat mass change pre to post pistachio enriched diet (PE) and pre to post no-pistachio control diet (NP) ...... 63

Figure 6 Pre and post 10 week pistachio (PE) treatment values for (a) LDL-C (b) T-C (c) T-C/HDL-C ratio and (d) LDL-C/HDL-C (ratio) ...... 66

Figure 7 Total cholesterol change pre to post pistachio enriched diet (PE) and pre to post no-pistachio control diet (NP) ...... 67

Figure 8 HDL cholesterol change pre to post pistachio enriched diet (PE) and pre to post no-pistachio control diet (NP) ...... 68

Figure 9 LDL cholesterol change pre to post pistachio enriched diet (PE) and pre to post no-pistachio control diet (NP) ...... 69

Figure 10 Triacylglycerol change pre to post pistachio enriched diet (PE) and pre to post no-pistachio control diet (NP) ...... 70

CHAPTER 1

INTRODUCTION

Statement of the Problem

According to the Centers for Disease Control’s (CDC) National Vital Statistics

Report, cardiovascular diseases are the leading cause of death in the United States

(Murphy, Xu, & Kochanek, 2013). The percentage of death from cardiovascular diseases

(CVD) in those with an optimal risk factor profile is 4.7% in men and 6.4% in women, whereas the percentage death from cardiovascular diseases in those with two or more major risk factors is 29.6% in men and 20.5% in women (Berry, Dyer, Cai, Garside, &

Ning, 2012). The risk factors used to determine this risk were: current smoking, diabetes, hypercholesterolemia, total cholesterol level of 240 mg per deciliter, hypertension, and systolic blood pressure of 160 mm Hg or diastolic blood pressure of 100mm Hg (Berry et al., 2012). An additional breakdown of lipid risk factors is detailed in the Third Report of the National Cholesterol Education Program, (NCEP, 2002) low density lipoproteins

(LDL-C) levels under 100 mg per deciliter are optimal for the prevention of CVD, 100-

129 mg/dl is near optimal, and higher numbers become a risk factor. This report also states that low high density lipoprotein (HDL-C), identified as fewer than 35 mg per deciliter, is an independent risk factor for CVD (NCEP, 2002).

Obesity and an increase in visceral fat have also been identified as risk factors for the development of CVD (Dudina, Cooney, Bacquer, Backer, & Ducimetière, 2011). It is estimated by the CDC that approximately one third of adults are obese, and one third of adults are overweight. Dietary intervention studies on weight loss are important, as are studies on dietary interventions that prevent obesity and overweight, as both reduce CVD

1 risk. There are strong correlations between CVD risk factors and death from CVD

(Dudina et al., 2011 Berry et al., 2012). Since major risk factors are correlated with death from CVD, ways to decrease these risk factors should be a major focus of public health studies.

Dietary interventions are a justified area of focus as they have been shown to be effective at reducing cardiovascular disease risk factors (Stone, Nicolosi, Kris-Etherton,

Ernst, & Krauss, 1996). Studies have examined the effects of specific eating patterns such as the “Western” or “Mediterranean” diet on the development of CVD risks and deaths

(Hodson, Skeaff, & Chisholm, 2001; Estruch, Martínez-González, Corella, Salas-

Salvadó, & Ruiz-Gutiérrez, 2006; Djuric, Ren, Blythe, VanLoon, & Sen, 2009; Carter,

Roberts, Salter, & Eaton, 2010; Estruch, Ros, Salas-Salvadó, Covas, & Corella, 2013).

Studies have investigated whole food interventions, meaning the examination of a specific food’s effects. Whole food studies are prevalent in research since the results can give insight into how specific foods could be healthfully added to a population’s diet to decrease, or simply not increase, CVD risk factors (Zambón, Sabaté, Muñoz, Campero, &

Casals, 2000; Chisholm, Mc Auley, Mann, Williams, & Skeaff, 2005).

Benefits of the Mediterranean Diet Pattern (MDP)

There is strong evidence that incorporating foods that are high in monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have a beneficial effect on blood lipid levels (Appel, Sacks, Carey, Obarzanek, & Swain, 2005;

Berglund, Lefevre, Ginsberg, Kris-Etherton, & Elmer, 2007). A diet high in MUFA and

PUFA, such as MDP, reduces blood cholesterol without an increase to triacylglycerols

(TAG), reducing CVD risk factors. Observational studies in Italy and the United States

2 found those who fell into the highest percentage of natural adherence to the

Mediterranean diet were less likely to be obese, diabetic, or hypertensive (Grosso, Pajak,

Mistretta, Marventano, & Raciti, 2014; Carter et al., 2010). A full conversion to the

Mediterranean diet pattern (MDP) may be hard as personality traits such as low self- directedness, and socio-demographic characteristics such as younger age and single marital status, have been shown to be significant factors in lower adherence to the

Mediterranean diet (Jurado, Burgos-Garrido, Diaz, Martínez-Ortega, & Gurpegui, 2012).

However, studies on specific aspects of the MDP have been shown to influence cardiovascular disease risk factors (Djuric et al., 2009).

The MDP is high in MUFAs and PUFAs, and replacing saturated fatty acid (SFA) intake with MUFA and PUFA intake has been shown to decrease total cholesterol and

LDL-C (Hodson et al., 2001). One common element of the MDP is the consumption of olive oil (Estruch et al., 2006). Olive oil is high in MUFA and is often used instead of solid fats, which are higher in SFA, when preparing food according to the MDP.

Consuming this high MUFA food over high SFA food may contribute to the CVD risk reduction (Djuric et al., 2009). Whole foods high in MUFAs and PUFAs while low in

SFA, such as tree nuts and avocado, have been targeted in dietary interventions (Wien,

Sabaté, Iklé, Cole, & Kandeel, 2003; Chisholm et al., 2005; Pieterse, Jerling, Oosthuizen,

Kruger, & Hanekom, 2005; Damasceno, Sala-Vila, Cofán, Pérez-Heras, & Fitó, 2013).

Nut Consumption and Dietary Fat

The data from the National Health and Nutrition Examination Survey 1999-2004, showed that the 6.5 percent of American adults who reported regularly eating nuts had a higher intake of MUFAs and PUFAs than non-consumers. Regular nut consumers also

3 had a 21% decrease risk of low HDL-C levels (O'Neil, Keast, Nicklas, & Fulgoni, 2012).

Research has indicated that nut consumption may lead to a reduction in cardiovascular disease risk factors and lower risk of all cause mortality (Bes-Rastrollo, Sabaté, Gómez-

Gracia, Alonso, & Martínez, 2007). Different nuts contain different percentages of

MUFA, PUFA, and SFA. Therefore, individual nuts are studies to understand their specific effects (Chisholm, Mann, Skeaff, Frampton, & Sutherland, 1998; Zambon et al.,

2000; Wien et al., 2003; Gebauer, West, Kay, Alaupovic, & Bagshaw, 2008).

Purpose of the Study

The purpose of this study is to examine the effects of regular pistachio consumption on blood lipid profiles, erythrocyte incorporation of fatty acids, and body composition on free-living healthy females, age 18-40. To measure effects on blood lipid profiles, collected and analyzed serum lipid levels and erythrocyte incorporation of fatty acids at baseline and post-intervention for each treatment period. To measure the effects on body composition, we measured and recorded body composition data at baseline and post-intervention for each treatment period. Additionally, we monitored compliance, and collected data on dietary quality, by having participants complete three sets of three-day

24-hour food records during each treatment period. This resulted in 18 collected 24 hour food logs for each participant by the end of the study.

Study Justification

This research will expand the scientific communities’ understanding of the effects of a whole food, the pistachio nut, on CVD risk factors. There is a current gap in the literature on how regular consumption of these nuts will affect healthy, free-living,

4 females. Our research is looking to advance the understanding of the effects of the pistachio nut while filling in the gap of research done on the healthy female population.

Statement of the Hypothesis

The aims of this study is to examine if daily consumption of pistachio nuts will help prevent or reduce the development of certain CVD risks in healthy women, age 18 to

40, without negatively affecting their body composition. All hypotheses are based on healthy females, with a BMI between 18.5 and 25, in which 20% of their daily calories are from pistachio nuts over a 10 week period.

Hypothesis 1

H0: Pistachio consumption will not change participant body weight

HA: Pistachio consumption will change participant body weight

Hypothesis 2

H0: Pistachio consumption will not change participant lean mass

HA: Pistachio consumption will change participant lean mass

Hypothesis 3

H0: Pistachio consumption will not change participant waist to hip ratio

HA: Pistachio consumption will change participant waist to hip ratio

Hypothesis 4

H0: Pistachio consumption will not change participant total cholesterol

HA: Pistachio consumption will change participant total cholesterol

Hypothesis 5

H0: Pistachio consumption will not change participant LDL-C

HA: Pistachio consumption will change participant LDL-C

Hypothesis 6

H0: Pistachio consumption will not change participant HDL-C

HA: Pistachio consumption will change participant HDL-C

Hypothesis 7

H0: Pistachio consumption will not change participant blood triacylglycerols

HA: Pistachio consumption will change participant blood triacylglycerols

Hypothesis 8

H0: Pistachio consumption will not change participant erythrocyte incorporation of fatty acids

HA: Pistachio consumption will change participant erythrocyte incorporation of fatty acids

Significance of the Study

Pistachio nuts have a particularly advantageous nutrient profile as one ounce of pistachio nuts is only 160 calories, with only 1.5g of SFA for the 3.8g of PUFA and 6.7g of MUFA (Dreher, 2012). Pistachios are also high in fiber at 2.8g per ounce, as well as potassium at 285mg. Pistachio nuts have a very high amount of lutein and zeaxanthin at

329 micrograms per ounce. However, many people still shy away from eating foods, such as nuts, due to their high fat content. Only 6.5 percent of Americans report eating nuts regularly (O’Neil et al., 2012). Many people may believe that nuts, due to their high fat content, will cause weight gain if regularly consumed. This study seeks to improve understanding of the role of pistachio nuts in a healthy diet. Currently, there is limited research on the effects of eating pistachios in a healthy female population. Since many females are concerned about weight gain, and the negative health effects that can come

6 with excess weight, it is important to examine if pistachio nuts can reduce cardiovascular disease risks by improving serum lipid profiles, without raising cardiovascular disease risks by gaining weight.

Definitions

Cardiovascular Disease (CVD). CVD is a class of diseases that centers on dysfunction of the heart and blood vessels. CVD manifests many problems with the heart and blood vessels. Many of these problems relate to atherosclerosis, a condition that develops as plaque builds up in, and narrows, the walls of the arteries. This narrowing restricts blood flow and increases the likely-hood that a blood clot could stop blood flow through the arteries, causing a heart attack or stroke (Dantas, Jiménez-Altayó, & Vila, 2012).

Low Density Lipoprotein (LDL). LDL is a class of lipoproteins responsible for the transport of cholesterol to extrahepatic tissues, including the arteries. LDL contains a relative abundance of cholesterol and, when deposited in the arteries, is associated with atherosclerosis. Decreasing serum LDL is the target of many CVD risk lowering interventions, and a decrease in LDL is a decrease in one CVD risk (NCEP, 2002).

High Density Lipoprotein (HDL). HDL is a class of lipoproteins that promote the transport of cholesterol from extrahepatic tissue to the liver for excretion in the bile. HDL is synthesized in the liver without a lipid core, as they travel from the liver and circulate through the bloodstream they pick up cholesterol for excretion. Raising serum HDL is a target of many CVD risk lowering interventions, however the therapies to elevate HDL have been inconsistent (NCEP, 2002).

Triacylglycerol (TAG). TAG is a compound consisting of three fatty acid molecules esterified to glycerol. A published Scientific Statement from the American Heart

Association states that “triglyceride is not directly atherogenic but represents an important bio-marker of CVD risk because of its association with atherogenic remnant particles and apo CIII (Miller, Stone, Ballantyne, Bittner, & Criqui, 2011).” Therefore an intervention which lowers TAG would be a positive intervention in the reduction of CVD risks.

Visceral Fat. Fat which accumulates in the intra-abdominal area is called visceral fat.

This type of fat is strongly associated with insulin resistance and with a typical atherogenic dyslipidemic state (Mathieu, Poirier, Pibarot, Lemieux, & Després, 2009).

High visceral fat is an independent risk factor for the development of cardiovascular disease (Dudina et al., 2011).

“Western” Pattern Diet. The Western Diet is a dietary intake usually high in SFA and refined carbohydrates. This diet is has reduced complex carbohydrate and fiber intake and reduced fruit and vegetable consumption (Francis & Stevenson, 2013).

“Mediterranean” Pattern Diet. The Mediterranean Diet is “characterized by high intake of olive oil, fruit, nuts, vegetables, and cereals” (Estruch et al., 2013). These are foods that are typically high in MUFA and low in SFA. The diet is also characterized by a low consumption of foods that are higher in SFA such as “dairy products, red meat, processed meats, and sweets” (Estruch et al., 2013). This diet has been shown to have cardio- protective effects by reducing the risks of CVD (Estruch et al., 2013; Grosso et al., 2013).

Free-living population. A free-living population is one in which participants will follow the eating pattern then did before the dietary intervention, with the only change being the specific intervention. Participants will eat their own food, with the exception of the provided intervention food, in their typical setting for the duration of the study. Free-

8 living participants are asked to maintain their current exercise and dietary patterns for the duration of the study. By having a free-living population to study we can maintain a high external validity as the intervention is implemented into the participant’s regular lifestyle.

Additionally, this requires less interference with normal life for the study participants since they are not required to eat meals under supervision.

CHAPTER 2

LITERATURE REVIEW

Current Dietary Recommendations to Lower the Risk of CVD

Dietary interventions are one of the available treatments to help reduce the risk of cardiovascular diseases. Different dietary interventions target different specific risks, such as high blood pressure, high total cholesterol, or overweight and obesity; while other interventions will target multiple risk factors.

The Adult Treatment Panel III Therapeutic Lifestyle Changes Diet

One commonly advocated dietary intervention for the reduction of cardiovascular diseases risk is the Therapeutic Lifestyle Changes (TLC) diet, also known as the Adult

Treatment Panel III (ATP III) diet, promoted by the National Cholesterol Education

Program (NCEP). The report identifies low density lipoprotein (LDL-C) as the primary target for this clinical cholesterol-lowering therapy. The NCEP advocates a two-pronged approach, population and clinical. The population dietary message advocates consuming low saturated fat foods, including reduced fat dairy products, leaner meats, lower fat ground meat, and reduced-fat baked goods. The clinical approach advocates for individualized diet counseling to promote the ATP III TLC diet, which is outlined in

Table 1. The final report includes a section on MUFAs, which outlines evidence that

MUFAs lower LDL cholesterol, relative to SFAs, and that MUFAs do not lower HDL cholesterol nor raise TAGs. This report recommends MUFAs as one replacement for

SFAs but notes that the benefits of replacing SFAs with MUFAs has not been adequately tested in controlled clinical trials. The recommendation notes that MUFAs should come from vegetable sources, including plant oils and nuts.

A retrospective observational study of patient compliance with the ATP III TLC diet showed that 44%-83% of patients adhered to the diet while attending the clinic for one year, but only 22%-31% complied with ATP III TLC diet follow up recommendations (Vijayakrishnan, Kalyatanda, Srinivasan, & Abraham, 2013). The conclusion was that this adherence was sub optimal. Therefore, while the diet may be effective, adherence to the diet is still an identifiable barrier to reducing CVD risks.

Table 1

Composition of the ATP III TLC Diet

Nutrient Recommended Intake

Polyunsaturated fat Up to 10% of total calories

Monounsaturated fat Up to 20% of total calories

Saturated fat <7% of total calories

Total fat 25-35% of total calories

Carbohydrate 50-60% of total calories

Fiber 20-30 grams per day

Protein Approximately 15% of total calories

Cholesterol <200 mg/d

The Dietary Approaches to Stop Hypertension (DASH) Eating Plan

The DASH diet reduces cardiovascular disease risks by lowering blood pressure.

The diet focuses on reduction of sodium intake, keeping it between 1,500 and 2,300 mg per day. The DASH eating plan also includes dietary recommendations such as low saturated fat and cholesterol. However, the diet plan makes no recommendations on

MUFA or PUFA dietary intake. The details of this eating plan are shown in Table 2.

Food specific recommendations provided to help patients follow the DASH diet include the recommendation to eat four to five servings of nuts, seeds, and legumes per week.

One serving is one and a half ounces nuts or two tablespoons of peanut butter.

Studies have shown that the DASH diet, in addition to lowering blood pressure, is also effective in lowering LDL cholesterol. A randomized controlled outpatient feeding study with subjects following the DASH diet showed that the diet resulted in lower total cholesterol, lower LDL cholesterol and lower HDL cholesterol, without any significant effects on TAG. The researchers concluded that the DASH diet may help reduce coronary heart disease risk, but there is a possible opposing effect with the reduction in HDL cholesterol, and therefore, further studies are warranted (Obarzanek, Sacks, Vollmer,

Bray, & Miller, 2001).

Table 2

Composition of the DASH Diet

Nutrient Recommended Intake

Total fat 27% of calories

Saturated fat 6% of calories

Protein 18% of calories

Carbohydrate 55% of calories

Cholesterol 150 mg

Sodium 2,300 mg

Potassium 4,700 mg

Calcium 1,250 mg

Magnesium 500 mg

Fiber 30 g

American Heart Association Guidelines for Women to Lower CVD Risks

The current effectiveness-based guidelines for the prevention of cardiovascular disease in women, published by the American Heart Association (AHA), include specific dietary intake recommendations for women. The general guidelines for dietary intake state that women should consume a diet rich in fruits and vegetables; to choose whole grains, high-fiber foods; to consume fish, especially oily fish at least twice a week; to limit intake of saturated fat, cholesterol, alcohol, sodium and sugar; and avoid trans-fatty

13 acids. These specific guidelines are shown in Table 3. The guidelines make no specific recommendations as to MUFA or PUFA intake. The specific guidelines include a recommendation for women to have four or more servings of nuts, seeds, or legumes per week. Serving is defined as 1/3 cups of 1 ½ oz nuts (avoid macadamia nuts and salted nuts), 2 tablespoons peanut butter, 2 tablespoons of ½ oz seeds, ½ cup cooked legumes

(dry beans and peas).

Table 3

Guideline from the AHA - Specific Dietary Intake Recommendations for Women

Nutrient Serving

Fruits and vegetables ≥4.5 cups/d

Fish 2/wk

Fiber 30 g/d (1.1 g/10 g carbohydrate)

Whole grains 3/d

Sugar ≤5/wk (≤450 kcal/wk from sugar-sweetened beverages)

Nuts, legumes, and seeds ≥4/wk

Saturated fat <7% total energy intake

Cholesterol <150 mg/d

Alcohol ≤1/d

Sodium <1500 mg/d

Trans-fatty acids 0

Dietary Guidelines for Americans

The Dietary Guidelines for Americans, 2010 are intended for Americans ages two years and older, including those at increased risk of chronic disease (McGuire, 2011).

Macronutrient amount recommendations for this diet are based off of the Dietary

Reference Intakes (DRI) and are shown in detail in Table 4. These guidelines recommend that adults 19 years and older consume 20-35% of total calories from fat, but to consume less than ten percent of calories from SFAs. The recommendation is that the rest of the fat consumed is from MUFAs or PUFAs, which are associated with low blood cholesterol levels, and therefore a lower risk of CVD. The guidelines note that lowering saturated fat intake to seven percent of calories can even further reduce the risk of cardiovascular disease.

Additionally, these guidelines note moderate evidence that higher intake of cholesterol may result in higher risk of cardiovascular disease. The guidelines advocate consuming less than 300 mg per day of cholesterol to maintain normal blood cholesterol levels; and consuming less than 200 mg per day to decrease CVD risk in individuals at high risk of CVD.

Table 4

Dietary Guidelines for Americans – Macronutrient Recommendations – Females 19-50

Macronutrient Recommended Intake

Protein 10-35% of calories

Carbohydrate 45-65% of calories

Total fiber 28 g/d age 19-30; 25 g/d age 31-50

Total fat 20-35% of calories

Saturated fat <10% of calories

Linoleic acid 12 g/d

Alpha-Linolenic acid 1.1 g/d

Cholesterol <300 mg/d

Potential Benefit Replacing SFA with MUFA and PUFA on CVD Risks

Of the four common dietary intake recommendations reviewed above, only two diets mention MUFA or PUFA intake. The ATP III TLC diet recommends that MUFA should be consumed as a replacement of SFA. The Dietary Guidelines for Americans recommends that 25-35% of the diet should come from fat, and of that only ten percent from SFA and the rest from MUFA and PUFA. The DASH diet plan and the AHA’s

“Guidelines for Women to Lower their CVD Risks” do not specifically mention MUFA or PUFA intake. However, recent scientific evidence shows that incorporating more

MUFA and PUFA into the diet can have beneficial results on serum lipids (Appel et al.,

2005; Mozaffarian, Micha, & Wallace, 2010).

In 1999, the AHA published a report on the relationship between MUFAs and

CVD (Kris-Etherton, Pearson, Wan, Hargrove, & Moriarty, 1999). This position paper reviewed studies that found when SFA was replaced by MUFA or carbohydrate (CHO) that LDL-C was significantly lower. However, only the replacement with MUFA did not lower HDL-C and did not increase TAGs as the CHO diet did (Kris-Etherton et al.,

1999). Since the AHA report, many studies found similar effects when a diet high in

MUFA was compared to a diet high in CHO (Appel et al., 2005) Other studies have found a reduction in LDL-C with both diets, and that the high CHO and high MUFA diets lower HDL-C, but the high CHO lowers the HDL-C significantly more than the high MUFA diet (Berglund et al., 2007). Meta-analysis of randomized controlled trials and cohort studies on MUFA and CVD risk reported that several studies showed an increase of HDL-C and a decrease in TAG after a MUFA rich diet, lowered LDL-C was inconsistent (Schwingshackl & Hoffmann, 2012). A study on young adults compared an intervention of higher MUFA versus higher PUFA intake in replacement of SFA. This study found that replacing SFA with PUFA or MUFA both significantly lowered LDL-C, but the diet high in MUFA did not significantly lower HDL-C, as the high PUFA diet did.

The MUFA and PUFA interventions both resulted in a lower T-C/HDL-C ratio (Hodson et al., 2001). Meta-analyses of randomized controlled studies on replacing SFA with

PUFA found that consuming PUFA over SFA reduces coronary heart disease events

(Mozaffarian et al., 2010). While the evidence for a reduction in cardiovascular disease risks from replacing SFA with higher MUFA and PUFA intake is strong, more free-living controlled studies need to be conducted to determine if this dietary pattern should be recommended for therapeutic or cardiovascular disease risk factor reduction therapy.

Pistachio Nuts Fatty Acid Composition

One ounce of pistachio nuts contains 7g of MUFA, 4g of PUFA, and 1.5g of SFA

(Dreher et al., 2012). Different fats have chemical differences: MUFAs have just one double bond, whereas PUFAs have two or more double bonds, SFAs have no double bonds. Fatty acids are almost completely absorbed by the intestine, and are necessary for the absorption of the fat soluble vitamins. After fats are absorbed, they can be broken down and used for energy, converted to other types of fatty acids, or incorporated into tissue lipids (Schwingshackl, & Hoffmann, 2012).

MUFAs are further divided by the configuration of their hydrogen atoms around the double bond. A cis configuration occurs when hydrogen atoms are on the same side of the double bond, whereas the trans configuration occurs when hydrogen atoms are on the opposite sides of the double bond. The cis configuration is the more common configuration in dietary fat intake, and for the purpose of this review, will be the configuration referred to whenever MUFA is referenced. The trans configuration will be referenced as trans-fats. The configuration of a trans-fat is linear, and therefore behaves more like a saturated fat. The cis configuration contains hydrogen atoms on the same side of the double bond and creates a kink in the fatty acid. Pistachio nuts naturally have no trans-fats (Dreher et al., 2010).

PUFAs have two or more double bonds. PUFAs have alpha-linolenic acid and linoleic acid, the essential fatty acids. The human body cannot synthesize essential fatty acids on its own. Alpha-linolenic acid (an omega-3 fatty acid) can be converted in to eicosapentaenoic acid (EPA) and docosahexaenonic acid (DHA) by enzymes in the human body. Linoleic acid is an omega-6 fatty acid; which converts to arachadonic acid

(AA) and other fatty acids needed in the formation of pro-inflammatory and anti- inflammatory molecules (Simopoulos, 2003).

By definition,SFAs have no double bonds and come in varying length depending on the number of carbon atoms. SFA is also referred to as the solid fat, as it is solid at room temperature due to its chemical structure (Sari, Baltaci, Bagci, Davutoglu, & Erel,

2010).

The chemical differences noted above cause each type of fat to behave differently in the human body. Thus, the different behavior of these fats is reflected in the studies on replacing SFA with MUFA and PUFA.

Effects of Nut Consumption on Serum Lipids and Weight Management

Large cohort studies have found that regular nut consuming has a positive impact on lipid profiles and weight management (Bas-Rastrollo et al., 2007; Martínez-González,

& Bes-Rastrollo, 2011; Casas-Agustench, Bulló, Ros, Basora, & Salas-Salvadó, 2011;

Casas-Agustench, López-Uriarte, Bulló, Ros, & Cabré-Vila, 2011; O’Neil et al., 2012;

Damasceno et al., 2013) The data, collected from NHANES, concluded consumption of nuts was associated with higher quality diet and a decrease in cardiovascular disease risk factors. Adult nut consumers had higher HDL-C, reducing their risk of CVD (O’Neil et al., 2012). Long term epidemiological studies found that nut consumption is not associated with higher risk of weight gain (Martinez-Gonzalez et al., 2010). The 5-year follow-up SUN project found that participants who consumed nuts regularly had a 56% lower risk of all-cause mortality than those who never, or almost never, consumed nuts

(Fernández-Montero, Bes-Rastrollo, Barrio-López, de la Fuente-Arrillaga, Salas-Salvadó,

2014).

Experimental studies, in nut interventions, have found that nut consumption lead to a more favorable lipid profile and no significant increase in weight (Zambon et al.,

2000; Wien et al., 2003; Ros, Núñez, Pérez-Heras, Serra, & Gilabert, 2004; Chisholm et al., 2005; Burns-Whitmore, Haddad, Sabaté, & Rajaram, 2014). Nuts have also shown to improve lipid profiles over other dietary interventions. When almonds were compared to complex carbohydrates in a weight reduction program, investigators found that almonds were associated with a greater reduction in weight, waist circumference, fat mass, total body water, and systolic blood pressure (Wein et al., 2003). In randomized cross over feeding trials on hypercholesterolemic participants, walnuts reduced total cholesterol and

LDL-C in comparison to the Mediterranean Diet alone and a diet high in MUFAs.

(Zambon et al., 2000; Ros et al., 2004). Therefore, it has been shown in epidemiological and experimental research that regular consumption of nuts is associated with a more beneficial lipid profile and without weight gain, in a variety of populations.

Pistachio Consumption and Serum Lipids

Studies on the effects of pistachio nut consumption looked at participants with higher cardiovascular disease risk, such as high LDL-C, moderate hypercholesterolemia, or those with metabolic syndrome (Edwards, Kwaw, Matud, & Kurtz, 1999; Sheridan,

Cooper, Erario, & Cheifetz, 2007, Gebauer et al., 2008; Gulati, Misra, Pandey, Bhatt, &

Saluja, 2014). These studies all showed an improvement in blood lipid profiles in some way, which decreases CVD risk. However, all but one of these studies’ intervention periods was equal to or less than four weeks of pistachio nuts consumption. Previous studies have shown serum lipids stabilize after five to eight weeks, therefore

20 interventions of three to four weeks may not be adequate to show a stable-change

(Ginsberg, Kris-Etherton, Dennis, Elmer, & Ershow, 1998).

Dose response studies of pistachio nuts were completed in participants with high

LDL, as well as in the healthy adult population (Gebauer et al., 2008). In the population with high LDL, three daily doses of pistachio nuts were tested. In a randomized cross over study participants consumed 0, 10, or 20% of their daily calories as pistachio nuts. It was shown the impact the nuts had on lipid profiles was dose dependent, and that 20% was the most effective dose (Gebauer et al., 2008). Three different doses were studied, utilizing a parallel design, in healthy adults and the doses were 0, 1.5, or 3 ounces of pistachio nuts daily. Gebauer (2008) found significant difference between pistachio consumption and non-consumption, but did not find a significant difference between the two doses. They concluded that for healthy adults consuming as low as 1.5 ounces a day could improve blood lipid profiles (Baer, Gebauer, & Novotny, 2012). Further studies need to be conducted to determine the effectiveness of dietary intake percentage versus a set dose of ounces per day of pistachio and the effects on blood lipids.

Nut Consumption and Erythrocyte Incorporation of Fatty Acids

Erythrocyte incorporation of fatty acids is a strong assessment of long-term fatty acid status, when compared to plasma lipid levels. The peak incorporation of fatty acids into erythrocyte membranes has been studied following fish oil ingestion. The study of fish oil showed peak incorporation at 180 days, with a half life between 27.7 and 36.7 days (Katan, Deslypere, van Birgelen, Penders, & Zegwaard, 1997). A study compared walnut consumption with a control diet and examined the effect on serum cholesterol esters fatty acid composition (Sabaté, Fraser, Burke, Knutsen, & Bennett, 1993). Sabaté

(1993) study showed higher levels of PUFA in serum cholesterol esters after walnut consumption. The fatty acid composition of walnuts is 10.1% SFA, 16.9% MUFA, 59.6%

PUFA, and 13.4% n-3 PUFA (ALA) (Ros et al., 2004). The high percentage of PUFA in walnuts may be responsible for the higher level of PUFA incorporation into serum cholesterol esters. There are currently no studies on pistachio nut consumption and how it may affect erythrocyte incorporation of fatty acids.

Pistachio Consumption and Weight Management

Most studies done on the effects of pistachio consumption included a weight or body composition measurement, taken at baseline and post-intervention. Studies with free living participants with both healthy and elevated CVD risk showed that consuming pistachio nuts regularly had no effect on body weight or BMI (Edwards et al., 1999;

Kocyigit, Koylu, & Keles, 2006; Gebauer et al., 2008; Sari et al., 2010; Gulati et al.,

2014). One study showed a significant decrease in waist circumference in those who consumed pistachios regularly (Gulati et al., 2014).

CHAPTER 3

METHODOLOGY

Participants

This research protocol, and all forms utilized in recruitment, screening, and selection, was approved by the Institutional Review Board (IRB) at California State

Polytechnic University, Pomona (CPP) (Protocol 12-139). The IRB approval letter is included as Appendix A.

The study participants were recruited from CPP. Recruitment procedure and protocols were developed in June 2012, and participants were recruited primarily via e- mail, as well as peer referral from initial interested students. Appendix B contains the sample e-mail.

The research objective was to examine the effects of a pistachio enhanced diet on healthy free-living women, our research sample of CPP students and staff were selected for convenience and their relatively low frequency of chronic diseases.

Screening

As part of recruitment, participants were asked to contact, in person or via e-mail, the campus primary investigator or graduate assistant. After this contact, interested women were asked to read and complete an Informed Consent form which described the purpose of the study, study procedure, inclusion and exclusion criteria, potential risks stress or discomfort, benefits of this study, what participation in the study entails, confidentiality information, and the experimental research subject’s bill of rights. The

Informed Consent Form can be found in Appendix C.

In addition to an Informed Consent Form, interested women were also sent the

Pistachio Study Initial Screening Questionnaire. This initial screening questionnaire can be found in Appendix D. This form allowed researchers to better assess if an individual would be a qualified candidate for the study. The form included questions on basic personal health and diet information, including whether or not the participant has or had any nut allergies and if they liked pistachio nuts. Participants were notified via e-mail if they qualified for the study based on their responses to the screening questionnaire.

Qualified participants were asked to schedule and initial visit to complete the screening process and begin the experimental period for those who were both qualified and interested.

Inclusion Criteria

In order to be eligible for this study, participants had to be students or staff at

CPP, biologically female, between ages 18 and 40, at a body mass index (BMI) between

18.5 and 25, and free of chronic diseases. Additionally, participants were asked if that liked pistachio nuts.

Exclusion Criteria

Persons were not eligible for this study, if they were not a student or staff at CPP, if they were not biologically female, if they were less than 18 years old or older than 40 years old, or had a BMI less than 18.5 or greater than 25. Interested persons were not eligible if they were allergic to pistachios or other nuts or if they ate an excessive amount of nuts. Additionally, if the interested person had a disease or chronic condition that required treatment that may affect metabolism, dietary intake, or physical activity levels, they were not eligible. The use of medications that affect fat malabsorption, laxative use,

32 liver disease, kidney disease, thyroid disease, diabetes, hypertension, dyslipidemia, pregnancy, intent to become pregnant, excessive use of alcohol, chronic disease, cancer, pacemakers or metal pins, plates in the body, or unwillingness to follow the study protocol which involves not consuming other nuts, fish or flaxseed oil or supplements, excluded interested persons from participating in the study. Lastly, interested persons who were elite athletes were also excluded from participating in this study.

Selection

Those women who were interested and passed the initial screening process came in for an office visit. Participants were asked to fast for 12 hours before this office visit, since they would have a fasting blood draw done that day if they enrolled in the study. At this visit, those who confirmed they were in good health, confirmed they were willing and able to adhere to the study design and agreed to maintain their current exercise routine and not take supplements throughout the duration of the study. After enrollment, those in the study completed the initial data collection visit.

Incentives

Participation in this study entailed blood draws and body composition measurements at baseline and post-intervention of each intervention period, resulting in four total measurement visits. At the end of this study, participants were told that they would have access to this information and could copy their lab results for their personal health files. Student participants were allowed to access to their blood draw lab results at any time. Participants received free pistachios while on the pistachio treatment and an additional bag of pistachio nuts at the end of their treatment period. Participants were compensated with $10.00 in “Bronco Bucks” at the end of the Fall 2012 quarter;

33 participants received an additional $10.00 in “Bronco Bucks” at the end of the Spring

2013 quarter. Participants that finished only one quarter were compensated with $10.00 in “Bronco Bucks”.

Participant Documentation

All documentation related to participants, including, but not limited to, their body composition data, blood work lab results, 24-hour dietary recalls, satiety data, physical activity and initial health screening questionnaires, and study related notes were coded with a study identification number (SID) were kept in a file coded with the SID and without the participant’s name. A master copy of the SID decoding sheet was kept separate to the coded data by the primary investigator and graduate assistant. The published results will exclude participant names and any personal information.

The primary investigator secured participant consent forms in a locked box for the duration of the study. The primary investigator held all raw data in a locked box after the study is complete until publication, after which the primary investigator will destroy the raw data sheets.

Experimental Design

The primary research objectives of this study were to examine the effects of a diet enriched with pistachios on body composition, red blood cell membrane incorporation of healthy fats, and changes to blood lipids in healthy free living women. Additionally, data was collected to assess the diet and macronutrient quality and satiety as a result of a pistachio enriched diet. Diet quality and satiety data will be discussed in another paper. In order to achieve adequate power (80%) to detect individual changes, a sample of 30 participants was used.

To examine the above effects, this study employed a cross-over study design. In this design, participants participated in each of the following interventions 1)No-pistachio diet(NP), in which participants abstained from eating pistachio or other nuts and 2)

Pistachio-enriched diet (PE), in which participants consumed pistachios nuts daily, in a provided amount which equaled 20% of their daily calorie intake. Participants were asked not to consume other nuts. Each dietary treatment would by 10 weeks long, which was determined as sufficiently long for the stabilization on serum lipids (Ginsberg at al.,

1998; Kris-Etherton et al., 1999). All participants completed a 15-week washout period in between treatment periods, during which participants consumed their regular diets.

Fasting blood draws and body composition analyses were completed at baseline and post- intervention of each treatment period. Figure 1 illustrates the study design.

Treatment Period 1 Washout Period Treatment Period 2 10 weeks 15 weeks 10 weeks * Pistachio Enriched * * No Pistachio Control * (3) 3-day diet records (3) 3-day diet records * No Pistachio Control * * Pistachio Enriched * (3) 3-day diet records (3) 3-day diet records

Figure 1. Summary of the crossover design employed in this study. The * represents blood draw and measurement lab visits. Light gray: Pistachio enriched treatment; White: No Pistachio control treatment; Dark gray: No treatment.

Participant Background Diet

All participants were screened to assure they were not following specific diets before enrolling in the study. Additionally, participants were screened to assure they were not consuming supplements regularly. Participants were free-living and were asked to maintain their background diet and only alter their intake of pistachio nuts, dependent on

35 their treatment group. Altering only pistachio intake, and keeping participants free-living, improved the external validity of this study.

Participant Background Exercise Routine

All enrolled participants completed the International Physical Activity

Questionnaire from August 2002 – Short Last 7 Days Self-Administered Format (IPAQ) form. This form was used first to confirm participant eligibility. This study excluded those who were elite athletes and the IPAQ allowed us to confirm non elite athlete status.

Additionally, this form was also used to determine activity level to be entered into the

Harris-Benedict equation, which determined the amount of calories a participant required per day to maintain their current weight. Finally, this form was used to monitor adherence to current exercise routine as it was administered before each treatment period.

Participants were asked to maintain their activity level throughout the duration of this study; IPAQ was used to confirm participants were maintaining their activity level. IPAQ form is provided in Appendix E.

Randomization to Dietary Treatments

All participants were randomized into one of two treatment groups upon enrollment in the study. Randomization was computer-generated, resulting in treatment groups ordered in a random sequence. Participants were informed of their condition after completing the screening and consent process. No participant objected to their initial assignment and all participants were made aware they would complete both interventions by the end of the study.

Pistachios

The pistachios for this study were provided by the American Pistachio Growers.

The pistachios were shipped to the CPP campus and picked up by study personnel.

Pistachios were stored in the plastic bag and cardboard boxes they were shipped in. These boxes were stored at room temperature. Nuts were checked for freshness by study personnel as they were measured out for participants to pick up. Study personnel wore latex gloves and a clean lab coat when handling pistachios. A food scale was used to measure out the required ounces of pistachio nuts to the nearest gram. Pistachios were then put into fresh plastic bags. Participants were given their week supply of pistachios in plastic bags which were put into a brown paper bag to help maintain freshness.

Participants received two weeks of pistachios during their first week in case they could not make it to pick up during one week of the study.

Pistachio-free diet

The pistachio-free diet was employed by all participants during one of the two treatment periods. During the pistachio-free diet, participants were asked to refrain from eating any pistachio nuts, as well as any other nut, nut butter, nut oil, or nut products. The diet required participants to maintain their normal eating pattern other than the avoidance of pistachios and other nuts or nut products.

Pistachio-enriched diet

The pistachio-enriched diet was employed by all participants during one of the two treatment periods. During the pistachio-enriched diet, participants were asked to eat

20% of their daily calories from the provided pistachio nuts. To determine 20% of daily calories, the Harris-Benedict equation was used. This equation provided the amount of

37 calories that would make up 20% of the diet. The number of calories was converted into grams of pistachio nuts. Pistachio nuts were measured out to the grams required for each participant in the treatment and were provided in individual plastic bags for daily consumption. Participants were asked to refrain from eating any other nuts, nut butters, nut oil, or nut products for the duration of this treatment period.

Adherence

Adherence to the diet was measured by asked participants to return all plastic bags each week when picking up their next weekly supply of pistachios. Adherence was also measured by participants completing 24-hour records and the unusual diet diary, provided in Appendix G.

Data Collection

In-person data collections occurred four times throughout the duration of the study. These collections occurred at baseline and post-intervention for each treatment period. Additional data collections of 24-hour dietary records were randomized into three sets of three-day 24-hour dietary records throughout each treatment period. Additionally, the unusual diet diary was provided to collect data for the full duration of each treatment period. A standardized data collection protocol was used for each participants visit.

Check-In

Participants checked into the lab between 8:00AM and 12:30PM on in-person data collection days. Participants were asked to confirm their fasting status before the study personnel went forward with the data collection. If the participant did not fast they were rescheduled for a future in-person data collection visit.

Height Procedure (Baseline visits only)

Participants were asked to remove their shoes and socks and step over to the stadiometer. Study personnel lifted the measuring device higher than the participant’s head and instructed the participant to step so their back was against the measuring tape.

Study personnel than lowered the measuring device until it just touched the highest point on the participant’s head. Study personnel then read and recorded the height in to 0.1cm on the participants Anthropometric Form, asked the participant to step forward. This procedure was then repeated. Height was taken at baseline visits only.

Blood Pressure Measurement

Participants were asked to sit quietly for three to five minutes before having blood pressure taken. Study personnel secured an automatic blood pressure monitor to the participants left wrist. Participant was instructed to place their feet flat on the floor and rest their left hand across their chest, according to the blood pressure monitors instructions. Study personnel turned on the automatic monitor and participant sat quietly while the cuff inflated then slowly deflated. Study personnel removed the monitor and recorded the blood pressure data on the Anthropometric Form. This procedure was repeated after one to two minutes of rest.

Waist-to-Hip Measurement

Participants had their waist and hips measured according to the standard protocol provided to the staff in the form of videos from the NHANES III Anthropometric

Procedure Videos. This protocol calls for measurements of the participant’s waist circumference to be taken with a flexible measuring tape placed between the participant’s middle of the bottom rib and iliac crest. This protocol calls for measurements of the

39 participant’s hips to be taken with a flexible measuring tape placed around the largest part of the hip or buttocks. These measurements were taken according to protocol and recorded to the nearest 0.1cm. Each measurement was taken 2 times during each data collection visit.

Tanita Bio-Impedance Scale Procedure

The body composition of each participant was measured at baseline and post- intervention of each treatment period. Body composition was measured using a Tanita

Bio-Impedance scale, model TBF-310GS. The Tanita scale utilizes an undetectable electrical current which passes through the participant’s body to provide a proxy for body composition. The current travels at different speeds when it is passing through lean muscle, which is high in water, as opposed to fat mass, which is lower in water and offers greater resistance to the current. Since the scale operated based off of conductivity and water, participants were asked to fast, and drink only 1 cup of water in the 12 hours before their data collection visit to ensure proper body water and provide for easy blood draws.

The Tanita scale was used to determine weight, BMI, percent body fat, impedance, fat mass, fat-free mass, total body mass, and total body water. In order for the

Tanita scale to accurately measure these body composition measurement points, the researchers were required to enter in basic information about the participant. Information to be entered in was clothing, gender, body type, age, and height. Clothing was entered as light; all participants took off heavy outerwear before stepping on the scale. Gender was always entered as female as all participants in this study were biologically female. Body type was entered in as “adult” for all participants. Age was entered in as the age the

40 participant began the study; if a participant had a birthday during the study, the initial age was used during all data collection visits to keep the measurement standardized and to prevent any instrumentation. Height taken by study personnel using a stadiometer was entered into the Tanita scale; height stayed consistent for all participants for the duration of the study.

Participants were instructed to remove all metal, including belts and any piercings below their necks before body composition could be measured. After metal was removed, study personnel entered in the participant’s personal information and asked them to step barefoot from the clean dry flood mat onto the cleaned and dried Tanita scale for about one minute as the scale completed its measurements. The Tanita scale then provided a print-out of the body composition information and the participant was asked to step off the scale. The scale was then cleared, the participant’s information was entered back in by study personnel, and the participant was asked to step back on the Tanita scale. A second print out of body composition information was provided. Study personnel dated and stapled both body composition print-outs into the participant’s coded file.

Blood Sampling

Participants were escorted by study personnel from the lab where the in-person data collection was set up, to Cal Poly Pomona Student Health Services where the trained phlebotomist would drew the participant’s blood. Participants were provided with a juice and small snack then escorted back to the initial lab room set up for the in-person data collection after their blood draw.

The phlebotomist filled two SST gel separated 8ml tubes followed by one EDTA

7ml tube. The SST tubes were handled and processed by Quest laboratories. The EDTA

41 tube was centrifuged and refrigerated by the phlebotomist at student health services before it was picked up by study personnel who extracted red blood cells to be sent to

Lipometrics laboratory for erythrocyte membrane analysis.

Blood Handling

After participant left the lab a sterile, polypropylene, conical-bottom 15 mL centrifuge tube and a sterile micro centrifuge tube were label for each participant with their SID and visit number. Approximately one hour after participants had their blood drawn; trained study personnel went to student health services to pick up a centrifuged and refrigerated EDTA 7 mL tube sample from each participant and brought them to the campus lab where the blood would be handled. The plasma, buffy coat, and uppermost erythrocytes were removed from the whole blood samples using a 200-1000 micro- pipette set to 100 micro-liters and discarded into a 1000 mL beaker containing a small amount of bleach. A 0.9% NaCl solution (saline solution) was added 1.0 mL at a time to help break up red blood cell clots to move liquid red blood cells by sterile pipette from the EDTA 7 mL tube into the labeled, sterile, polypropylene, conical-bottom 15 mL centrifuge tube. Red blood cells that had coagulated were stored at – 80 degrees Celsius in the original EDTA 7 mL tubes so study investigators would have access to original samples if needed. Red blood cells with the saline solution were transferred into the 15 mL centrifuge tube up to the 7.0 mL mark. The 15 mL tubes were then centrifuged at

2950 RPM/RCF at 4 degrees Celsius for 10 minutes, in a properly balanced centrifuge.

Following centrifuging surface layer of saline solution was removed using a 200-1000 micro-pipette set at 100 micro-liters and discarded in the 100 mL bleach containing beaker. Dark layer of red blood cells were left behind. Fresh saline solution was added to

42 the 15 mL centrifuge tube up to the 7.0 mL line. Tube was gently agitated and placed back in centrifuge for another 10 minutes, again at 2950 RPM/RCF at 4 degrees Celsius.

This centrifuging process was repeated one more time. After the third centrifuging process fresh saline was added to the red blood cells in a ratio of 2:1 red blood cells to saline solution. Two mL of the red blood cell and saline mixture was transferred into the labeled sterile micro centrifuge tubes. Remaining red blood cell solution was transferred in to a 5.0 mL test tube for back up storage. Original sample, micro-centrifuge tube, and

5.0 mL test tube were all stored in a designated deep freeze at -80 degrees Celsius.

There was a refrigeration malfunction on a Sunday during the study and all samples were relocated when the malfunction was discovered on the following Monday morning. Samples did not seem affected as they were relocated quickly. Samples were shipped frozen to Lipomics Technologies (West Sacramento, CA) for TrueMass Fatty

Acid Metabolism Panel. Results of this panel were send to study investigators in a

Microsoft excel file.

Dietary Counseling

Participants were taught to complete the 24-hour dietary records. They were given a hand out on common portion sizes to help them estimate portions when recording their food intake. Participants were instructed to complete their hunger/satiety rating using the

“Hunger/Satiety Scale” or (HSS). Participants were told to designate each meal a breakfast (B), lunch (L), dinner (D), or snack (S). They were instructed to record each food and beverage consumed, and to include brands when consuming brand named foods.

They were instructed to indicate the cooking method, including if any fat, salt, or seasoning was added to the dish. They were instructed to record the portion size, using

43 the common portion sizes handout to help them record amounts. Participants were instructed on how to use the visual analog scale (VAS) to also measure their hunger and satiety. Additionally, participants were shown how to use the Unusual Diet Diary to record abnormal eating or medication days, as well as any other days the participant did not comply with the study protocol, including if they accidentally ate nuts. Lastly, participants were counseled on how to maintain an isocaloric diet while consuming the pistachios, by identifying where on “MyPlate” the pistachios would fit into their eating pattern.

Participants were informed of, and reminded to record on, the dates they were expected to complete their 24-hour records which had been randomized into three sets of three-day 24-hour dietary records throughout the quarter.

Variation from Scheduled Intervals

The 10 week treatment periods for this study coincided with the California State

Polytechnic University, Pomona quarter duration which includes 10 class meeting weeks plus one final exam meeting week. In order to accommodate participant’s academic or work schedules participants were required to complete each treatment for a minimum of

10 weeks, but an additional grace period of 5 days was given to ensure that all participants were able to complete their fasting blood draws and body composition measurements. During this grace period, participants maintained their assigned treatment of NP or PE diets.

Data Analysis

Raw data were entered into Microsoft Office Excel 2007 spreadsheets. Body composition, serum lipids, and erythrocyte fatty acid composition taken before the pistachio treatment were entered in as variable X one, the post pistachio treatment values were entered in as variable X two, the body composition and blood values taken before the no pistachio control treatment were entered in as variable X three, the values taken post no pistachio control treatment were entered in as variable X four. Pre and post treatment differences were calculated by subtracting variable X one from variable X two

(XDIFF12), and variable X three from variable X four (XDIFF34). Differences were calculated for body composition, serum lipids, and erythrocyte fatty acid composition variables.

The data set created in Excel was imported into SPSS (version 22), for statistical analysis. Based on recommendations from Tabachnick & Fidell (2007) assumptions of normality were checked (Tabachnick & Fidell, 2007). Based on recommendations from

Morgan (2007) a comparison of means for the variables expressing the difference in treatments, “XDIFF12” and “XDIFF34”, was determined using a paired samples t test

(Morgan, Leech, Gloeckner, & Barrett, 2007).

CHAPTER 4

RESULTS

Participant Characteristics

Thirty-one women, recruited from CPP enrolled in the study. One participant had a negative reaction to having blood drawn and dropped out during the initial week of the study; the participant was replaced by the end of the first week, resulting in 30 enrolled participants. Of the 30 women recruited, seven women dropped out during the course of the study, leaving 23 participants (n=23), see Figure 2. The average participant baseline, pre-pistachio treatment, age was 22 ± 4 years with average BMI of 22 ± 2. The average participant total cholesterol (T-C) was 154 ± 32 with an LDL of 79 ± 26. The average participant erythrocyte %MUFA was 19 ± 4, %PUFA was 30 ± 7. Characteristics reported as pre pistachio treatment means ± standard deviation.

Participant baseline anthropometric characteristics are shown in Table 13.

Participant baseline serum lipid profiles are shown in Table 14. Participant baseline erythrocyte fatty acid percentages are shown in Table 15. Body composition and serum lipid values are available for the 23 participants. Fatty acid composition data pre and post pistachio treatment are available for 20 participants (n=20) and for 22 participants (n=22) pre and post no pistachio control treatment; four baseline samples from the first treatment period (Fall 2012) were not able to be analyzed due to equipment malfunction; these participants’ first treatment data was excluded from statistical analysis.

Figure 2. Summary of screening and enrollment demographics

Table 13

Mean Participant Baseline, Pre-pistachio Treatment, Anthropometric Characteristics

Standard

Measure Mean Deviation Minimum Maximum

Age (years) 22.8 4.45 18.0 33.0

Height (cm) 164 7.29 151 181

Weight (kg) 60.3 7.89 44.8 73.5

Body Mass Index (BMI) 22.3 2.20 18.2 27.3

Body Fat Percentage (BFP) 26.1 5.49 14.5 36.3

Fat Mass (kg) 16.1 5.29 6.50 26.7

Fat Free Mass (kg) 44.2 3.23 38.3 49.5

Body Water (kg) 32.4 2.36 28.1 36.3

Waist Circumference (cm) 28.6 1.89 25.2 32.0

Hip Circumference (cm) 39.0 2.02 35.5 43.3

Note. All baseline anthropometric values are reported from pre-pistachio treatment, obtained during week 0 prior to the start of 10-week pistachio treatment. n=23.

Table 14

Mean Participant Baseline, Pre-pistachio Treatment, Serum Lipid Profile

Standard

Measure Mean Deviation Minimum Maximum

LDL-C 79.0 26.1 20.0 140

HDL-C 60.0 13.7 23.0 83.0

T-C 154 32.4 79.0 223

TAG 74.3 34.7 37.0 161

T-C/HDL (ratio) 2.65 0.57 1.34 3.96

LDL-C/HDL-C (ratio) 1.38 .503 .240 2.64

Note. All baseline serum lipid values are reported from pre-pistachio treatment, obtained during week 0 prior to the start of 10-week pistachio treatment. n=23.

Table 15

Mean Participant Baseline, Pre-pistachio Treatment, Erythrocyte Fatty Acid Percentages

Standard

Measure Mean Deviation Minimum Maximum

% Saturated Fatty Acids (SFA) 45.7 4.50 37.8 55.1

% Monounsaturated

Fatty Acids (MUFA) 19.4 3.92 14.9 27.0

% Polyunsaturated

Fatty Acids (PUFA) 30.5 7.09 18.2 38.3

% Omega 3 Fatty Acids (n3) 4.45 1.92 1.16 7.52

% Omega 6 Fatty Acids (n6) 26.0 5.29 16.6 31.6

% Omega 7 Fatty Acids (n7) 5.06 3.31 1.49 11.7

% Omega 9 Fatty Acids (n9) 14.4 1.36 11.8 18.5

% Plasmologen-linked

Fatty Acids (dm) 4.44 2.06 1.88 8.02

Note. All values are represented as mole percentage of fatty acid in the erythrocyte membrane. n=20

Hypothesis 1

Null hypothesis 1 posits that consuming 20% of daily calories from pistachio nuts over a 10 week period (regular pistachio consumption) will not change body weight of healthy women between ages 18 and 40 (healthy participants). Results of a paired samples t test indicated that participants’ body weight did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = 1.20, p =

.24, d = .24. The difference is not statistically significant. Participant body weight did not significantly change pre and post pistachio treatment, t (22) = 1.51, p = .14, d = .32.

Therefore, Hypothesis 1 is supported.

Hypothesis 2

Null hypothesis 2 posits that regular pistachio consumption will not change lean mass of healthy participants. Results of a paired samples t test indicated that participants’ lean mass did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = .89, p = .381, d = .19. The difference is not statistically significant. Participant lean mass did not significantly change pre and post pistachio treatment, t (22) = -1.59, p = .13, d = -.33. Therefore, Hypothesis 2 is supported.

Hypothesis 3

Null hypothesis 3 posits that regular pistachio consumption will not change waist to hip ratio of healthy participants. Results of a paired samples t test indicated that participants’ waist to hip ratio did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = 1.20, p = .25, d = .25. The difference is not statistically significant. Participant waist to hip ratio did not significantly

51 change pre and post pistachio treatment t (22) = .89, p = .38, d .19. Therefore, Hypothesis

3 is supported.

Hypothesis 4

Hypothesis 4 posits that regular pistachio consumption will change total cholesterol of healthy participants. Results of a paired samples t test indicated that participant total cholesterol did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = -.40, p = .690, d = -.08.

The difference is not statistically significant. Participant total cholesterol did not significantly change pre and post pistachio treatment t (22) = -.56, p = .60, d -.11.

Therefore, Hypothesis 4 is not supported.

Hypothesis 5

Hypothesis 5 posits that regular pistachio consumption will change LDL-C of healthy participants. Results of a paired samples t test indicated that participants’ LDL-C did not significantly change during regular pistachio consumption, when compared to the no pistachio control, t (22) = -.75, p = .463, d = -.16. The difference is not statistically significant. Participant LDL-C did not significantly change pre and post pistachio treatment t (22) = -.70, p = .49, d -.15. Therefore, Hypothesis 5 is not supported.

Hypothesis 6

Hypothesis 6 posits that regular pistachio consumption will change HDL-C of healthy participants. Results of a paired samples t test indicated that participants’ HDL-C did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = -.03, p = .980, d = -.01. The difference is not statistically

52 significant. Participant HDL-C did not significantly change pre and post pistachio treatment t (22) = -.28, p = .78, d -.06. Therefore, Hypothesis 6 is not supported.

Hypothesis 7

Hypothesis 7 posits that regular pistachio consumption will change blood triacylglycerols of healthy participants. Results of a paired samples t test indicated that participants’ blood triacylglycerols did not significantly change during regular pistachio consumption, compared to no pistachio control, t (22) = 1.51, p = .145, d = .32. The difference is not statistically significant. Participant triacylglycerol did not significantly change pre and post pistachio treatment t (22) = .60, p = .55, d .13. Therefore,

Hypothesis 7 is not supported.

Hypothesis 8

Hypothesis 8 posits that regular pistachio consumption will change erythrocyte incorporation of fatty acids of healthy participants. Results of a paired samples t test indicated that participants’ erythrocyte incorporation of fatty acids did not significantly change during regular pistachio consumption, when compared to no pistachio control.

Erythrocyte incorporation of saturated fatty acids did not significantly change between the pistachio and control groups. However, mean percent polyunsaturated fatty acid, omega 6 fatty acid, omega 9 fatty acid and plasmologen-linked fatty acid erythrocyte composition increased in both the pistachio treatment and control groups, while mean percentage saturated fatty acid, monounsaturated fatty acid, omega 3 fatty acid and omega 7 fatty acid erythrocyte composition decreased in both the pistachio and control groups. There were no significant differences in the percent fatty acid change between the two groups. There was a decrease in percentage monounsaturated fatty acid during both

53 treatments, with a significant decrease following the pistachio treatment, PE: t (19) = -

2.33, p = .03, d = -.52. Significant decreases in omega 7 fatty acid composition were found during the pistachio and non pistachio treatment, PE: t (19) = -4.43, p = <.01, d = -

.99 and NP: t (21) = -3.56, p = <.01, d = -.76. There was a significant increase in plasmologen-linked fatty acids during the pistachio and non pistachio treatment, PE: t

(19) = 5.77, p = <.01, d = 1.29 and NP: t (21) = 5.25, p = <.01, d = 1.12 Therefore,

Hypothesis 8 is supported with a significant change in monounsaturated, omega 7, and plasmologen-linked fatty acid composition during the pistachio treatment.

CHAPTER 5

DISCUSSION

Research Basis

There is substantial evidence that consuming a diet that is high in MUFA while low in SFA can reduce cardiovascular disease risks (Berglund 2007; Schwingshackl, &

Hoffmann, 2012). However, the predominant eating pattern in the United States is the

“Western Pattern Diet,” characterized by high intake of SFA and refined carbohydrates

(Cordain, Eaton, Sebastian, Mann, & Lindeberg, 2005). According to a study done in

2012 conversion to a diet that is high in MUFA and low in SFA, such as the MDP, can be difficult (Jurado et al., 2012). Observational evidence shows that adults, who live in the

United States and consume nuts regularly, have a decreased risk of cardiovascular disease

(O’Neil et al., 2012). This evidence shows that those who ate nuts had better nutrient intake, diet quality, and health risk parameters than non-consumers. However, the evidence used was cross-sectional and, therefore, causal inferences cannot be drawn.

In this study we examined what changes occur when the diet of a healthy female is maintained, with the only change consisting of pistachio nut consumption. Pistachio nuts are high in MUFA and low in SFA, similar to the MDP. Other nuts high in MUFA and low in SFA, studied in controlled feeding studies, have been shown to reduce various cardiovascular disease risk factors in a variety of populations (Jenkins, Kendall, Marchie,

Parker, & Connelly, 2002; Wien et al., 2003; Chisholm, Mc Auley, Mann, Williams, &

Skeaff, 2005). Pistachio nuts have been studied in the following types of participants: participants with elevated LDL-C, participants with hypercholesterolemia, participants with metabolic syndrome, participant who consumed pistachio nuts in conjunction with,

55 or after consuming, a Mediterranean diet, and in participants who were healthy men and women (Edwards et al., 1999; Kocyiget et al., 2006; Sheridan et al., 2007; Gebauer et al.,

2008; Sari et al., 2010; Gulati et al., 2014). The effects of regular pistachio nut consumption on body composition, serum lipids, and erythrocyte incorporation of fatty acids, in healthy women had not been previously studied.

Research Participant Means

The participants in this study were free living females, recruited from CPP, between the ages of 18 and 40 at a BMI between 18.5 and 25. All participant means were in the AHA’s ideal BMI range of 18.5 and 25, as well as within the ideal waist circumference range of fewer than 88 cm or 35 in (Mosca, Benjamin, Berra, Bezanson, &

Dolor, 2011). Participant pre-pistachio enriched treatment (PRE-PE) and pre no pistachio control treatment (PRE-NP) means all fell within the ideal serum lipid ranges, as advocated for women by the AHA (Mosca et al., 2011); see table 16 for serum lipid reference ranges. A paired samples t test was performed on the pre-treatment values for each treatment condition; there was no significant difference in baseline between the groups (p < 0.05), as expected in a cross-over study with a proper washout period.

Participant post pistachio enriched treatment (POST-PE) and post no pistachio control treatment (POST-NP) means and ideal ranges are in table 17. Erythrocyte fatty acid composition does not have established reference ranges; therefore results are looked at by mean changes, and not compared to recommended reference ranges.

Table 16

Pre and Post PE Intervention Serum Lipid Means and AHA Advocated Serum Lipid

Values for Women

Participant Mean Participant Mean Ideal

Serum Lipid PRE-PE(SD) POST-PE(SD) Change P value Range

LDL-C (mg/dL) 79.0 (5.5) 6.70 (4.8) -2.30 .49 <100

HDL-C (mg/dL) 60.0 (2.9) 59.7 (2.6) -.348 .78 >50

T-C (mg/dL) 154 (6.7) 152 (6.1) -2.10 .59 <200

TAG (mg/dL) 74.3 (7.2) 76.8 (8.1) 2.52 .55 <150

T-C/HDL-C (ratio) 2.65 (.12) 2.60 (.11) -.043 .48 <5

LDL-C/HDL-C (ratio) 1.38 (.50) 1.33 (.45) -.050 .38 N/A

Note. All serum lipid values are shown as mg/dL with the exception of the T-C/HDL-C and LDL-C/HDL-C which are shown as ratios. Ratios were determined by dividing T-C by HDL-C and LDL-C by HDL-C. Participant pre pistachio (PE) treatment means are shown first, followed by post pistachio (PE) treatment means. This is followed by change, which was calculated by subtracting the pre pistachio treatment means from the post pistachio treatment means. P values were calculated using a paired samples t test. The ideal range of serum lipids is provided by the American Heart Association (Mosca et al., 2011). n=23.

Table 17

Pre and Post NP Intervention Serum Lipid Means and AHA Advocated Serum Lipid

Values for Women

Participant Mean Participant Mean Ideal

Serum Lipid PRE-NP (SD) POST-NP (SD) Change P value Range

LDL-C (mg/dL) 77.2 (4.8) 78.6 (5.1) 1.43 .66 <100

HDL-C (mg/dL) 62.0 (2.6) 61.7 (3.0) -.304 .83 >50

T-C (mg/dL) 155 (5.8) 155 (6.7) .130 .97 <200

TAG (mg/dL) 76.8 (6.4) 71.5 (6.0) -5.30 .99 <150

T-C/HDL-C (ratio) 2.57 (.12) 2.57 (.10) .001 .20 <5

LDL-C/HDL-C (ratio) 1.32 (.51) 1.33 (.43) .010 .38 N/A

Note. All serum lipid values are shown as mg/dL with the exception of the T-C/HDL-C and LDL-C/HDL-C which are shown as ratios. Ratios were determined by dividing T-C by HDL-C and LDL-C by HDL-C. Participant pre no pistachio (NP) treatment means are shown first, followed by post no pistachio (NP) treatment means. This is followed by change, which was calculated by subtracting the pre no pistachio treatment means from the post no pistachio treatment means. P values were calculated using a paired samples t test. The ideal range of serum lipids is provided by the American Heart Association (Mosca et al., 2011). n=23.

Body Composition

There were no significant results when comparing body composition changes during the PE diet to body composition changes during the NP diet. These results are similar to other nut consumption studies where participant diets were isocaloric and activity level was maintained (Chisholm et al., 1998; Zambon et al., 2000; Kocyigit et al.,

2006; Sari et al., 2010), and to a prospective study which looked at regular nut consumption and found it was not associated with weight gain (Bes Rastrollo et al.,

2007). Studies on nut consumption that have found a reduction in weight, BMI, or waist circumference also included a calorie reduction, or exercise component in the intervention (Wien et al., 2003; Li, Song, Nguyen, Zerlin, & Karp, 2010; Gulati et al.,

2014).

Both the PE diet and NP diet saw a significant increase in body fat percentage from the pre to post intervention, PE: t (22) = 2.82, p = .01, d = .59 and NP: t (22) = 2.15, p = .04, d = .45. Both the PE diet and NP diet saw a significant increase in fat mass from the pre to post intervention. Pistachio treatment increase was significant PE: t (22) = 3.06, p = <.01, d = .64. No pistachio control treatment approached significance NP: t (22) =

1.84, p = .08, d = .39. Change in fat free mass decreased pre to post treatment in both treatment groups; the decrease was significant in the no pistachio control treatment NP: t

(22) = -2.37, p = .03, d = -.49. The decrease was not significant in the pistachio treatment group PE: t (22) = -1.59, p = .13, d = -.33. The final variable with any significant change between PRE-PE and POST-PE or PRE-NP and POST-NP was in body water. Both groups decreased in body water. The PE diet decreased, but not significantly, PE: t (22) =

-1.93, p = .07, d = -.40. The NP diet decreased significantly NP: t (22) = -2.77, p = .01, d

= -.58. Figure 3 shows the mean changes pre and post PE and NP treatments. Participants had a wide range in body fat percentage as well as fat mass, the values pre and post each treatment for each participant is shown in Figure 4 and Figure 5, respectively.

The mean weight gain in both the PE and NP diet may be a reflection of the study length coinciding with the academic quarter. A study on weight gain in college freshman showed significant weight gain from September to December (67.1 ± 1.2 vs. 68.3 ± 1.2 kg, respectively; P <.01); with additional but non-significant weight gain from December to May (68.3 ± 1.2 vs. 69.7 ± 1.3 kg, respectively; P>.05) (Anderson, Shapiro, &

Lundgren, 2003). The weight gain study followed students on a semester system, which varies from the quarter system followed in the current study. Our study baseline measurements were taken in the beginning of a 10-week quarter, with a washout period of 15 weeks, which included a winter break when students were not in school. This break could be responsible for participants returning to their mean baseline body composition before the second treatment period. The post intervention measurements were taken during the finals week of each quarter. Therefore, the study results may reflect a trend of weight and body fat gain during a 10-week academic quarter. This conclusion is drawn since the mean gain was seen in both the PE and NP groups, and was independent of treatment order.

(a) (b)

27 16.8 26.8 16.6

26.6 16.4 26.4 Pre-treatment Pre-treatment 26.2 16.2 26 Post- MassFat 16 Post- Body Fat% Body 25.8 treatment treatment 25.6 15.8 25.4 15.6 PE diet NP diet PE diet NP diet

44.4 32.6

44.2 32.4

44 Pre-treatment 32.2 Pre-treatment

43.8 Post- 32 Post- Body WaterBody

Fat Free MassFat treatment treatment 43.6 31.8

43.4 31.6 PE diet NP diet PE diet NP diet

Figure 3. Pre and post 10 week pistachio enriched (PE) and no pistachio control (NP) diets. a. Body fat percentage b. Fat mass c. Fat free mass d. Body water. n=23.

Serum Lipids

Following the PE diet there were non-significant favorable mean changes in LDL-

C t (22) = -.70, p = .42, d = -.15 in T-C, t (22) = -.55, p = .59, d = -.11 in T-C/HDL-C, t

(22) = -.72, p = .48, d = -.15 and also in LDL-C/HDL-C, t (22) = -.90, p = .38, d = -.19.

Figure 4 show mean changes of the pistachio treatment on LDL-C, T-C, T-C/HDL-C and

LDL-C/HDL-C.

There were no significant serum lipid changes when comparing changes in serum lipids during the PE and NP treatments. Other studies have shown changes in serum lipids during pistachio treatments (Edwards et al., 1999; Kocyigit et al., 2006; Sheradin et al., 2007; Gebauer et al., 2008; Sari et al., 2010; Baer et al., 2012; Gulati et al., 2014).

Many of these studies were done in non-healthy participants, which include hypercholesterolemic participants, those with elevated LDL-C or those with metabolic syndrome (Edwards et al., 1999; Sheridan et al., 2007; Gebauer et al., 2008; Gulati et al.,

2014). Studying participants with an already elevated or modified serum lipid profile could be responsible for the difference in results to the current study on healthy normolipidemic participants. Individual participant changes in total cholesterol, HDL-C,

LDL-C and TAG pre to post PE and NP diets are shown in tables 7, 8, 9 and 10 respectively.

Other pistachio consumption studies done in healthy participants have shown a reduction in serum lipid levels, in contrast to what was seen in the current study (Kocyigit et al., 2006; Sari et al., 2010; Baer et al., 2012). Kocyigit (2006) completed a study on healthy individuals consuming 20 percent of their daily calories from pistachio nuts over three weeks and found a significant decrease in T-C, T-C/HDL-C, and LDL-C/HDL-C.

The Kocyigit (2006) study varied from the present study as participants in the pistachio group consumed reduced portions of fatty foods such as meat, and the amount of visible fats consumed was decreased to accommodate the percentage of energy from pistachio nuts. Therefore, the Kocyigit (2006) study results are more of a reflection of purposefully replacing saturated fats with the fats from pistachio nuts, as opposed to the current study which did not instruct participants to reduce their saturated fat intake. Sari (2010) found a significant reduction in LDL-C, T-C and TAG, however the baseline values for those in this study were at the high end of the ideal range: LDL-C mean 124.5, T-C mean 190.7, and TAG mean 112.1, and lowered down to the following means LDL-C mean 95, T-C mean 149.4 and TAG mean 91.1. The post intervention means in the Sari (2010) study were similar to, or still above the current studies baseline and post intervention means, see Table 16 and 17. Baer (2012) found a significant reduction in LDL-C when three varying pistachio doses were consumed for eighteen consecutive days. The study also found that dietary fat in pistachio nuts is poorly metabolized (Baer et al., 2012). The metabolisable energy (ME) value of pistachio nuts was found to be less than the Atwater general factors, which is how energy content of food is primarily determined.

Additionally, there was a notable inter-individual variability in ME values within the eighteen study participants. The current study did not examine the energy value of nuts and, therefore, we do not know how efficiently the examined participants metabolized the fats from the pistachio nuts.

(a) (b)

80 155 79 154 78

153 77 Pre-treatment Pre-treatment 152 76 mg/dL Post- mg/dL 151 Post- 75 treatment treatment 74 150 73 149 LDL-C T-C

2.68 1.42 2.66 1.4 1.38 2.64 1.36 Pre-treatment Pre-treatment 2.62 1.34 ratio

ratio 2.6 1.32 Post- Post- 2.58 1.3 treatment 1.28 treatment 2.56 1.26 2.54 LDL-C/HDL- T-C/HDL-C C

Figure 6. Pre and post 10-week pistachio (PE) treatment values for (a) LDL-C, (b) T-C (c) T-C/HDL-C ratio and (d) LDL-C/HDL-C ratio. Measurements taken at week 0 and week 10. Ratios calculated by dividing T-C by HDL-C and LDL-C by HDL-C. n=23

Erythrocyte incorporations of fatty acids

The regular consumption of pistachio nuts did not significantly alter the erythrocyte fatty acid composition when compared to the NP group. There was a significant decrease detected when a paired samples t test was run for percent MUFA

PRE-PE to POST-PE t (19) = -2.33, p = .03, d = -.52, see table 18 for PRE-PE to POST-

PE values. A retrospective power calculation was conducted for this variable due to the smaller than expected sample size as a result of damaged samples. The power of this calculation was found to be 52% when performing retrospective power calculations and, therefore, the t test result is not a statistically significant result. There was also a mean percent MUFA decrease that approached significance from PRE-NP to POST-NP t (21) =

-1.90, p = .07, d = -.40, see table 19 for PRE-NP to POST-NP changes.

Other studies have shown some significant change in erythrocyte fatty acid composition after a dietary intervention (Barceló et al., 2009; Rajaram, Haddad, Mejia, &

Sabaté, 2009; McKay, Chen, Yeum, Matthan, & Lichtenstein, 2010). However, Barceló

(2009) and Rajaram (2009) both studied the effects in hyperlipidemic participants

(Barceló, Perona, Prades, Funari, & Gomez-Gracia, 2009; Rajaram et al., 2009).

Therefore, those results are not applicable to our healthy normolipidemic participants.

The McKay (2010) study was done with a walnut intervention; walnuts and pistachio nuts have different dietary fatty acid compositions (McKay et al., 2010; Dreher 2012).

Other nut consumption studies have shown that there is inter-personal variety in the metabolism of dietary fat from nuts (Traoret, Lokko, Cruz, Oliveira, & Costa, 2008;

Baer et al., 2012; Novotny, Gebauer, & Baer, 2012). It was found that the Atwater factors of almonds and pistachios overestimated the amount of energy the nuts provided. For

71 almonds, the Atwater factors overestimated energy by 32% (Novotny et al., 2012). For pistachios, the Atwater factors overestimated energy by 5% (Baer et al., 2012).

Additionally, it is found that energy loss was greater when whole nuts were consumed as opposed to nut butters, oils, or flours (Traorat et al., 2008). The current study participants were provided with whole pistachio nuts, and most participants consumed them in their whole forms. The digestion and metabolism of these nuts could potentially have had an effect in the incorporation of fatty acids into the erythrocyte membrane.

Table 18

Pre and Post PE Intervention Percent Fatty Acid of Erythrocyte Membrane

Participant Mean Participant Mean

Erythrocyte Membrane PRE-PE (SD) POST-PE (SD) Change P value

% Saturated Fatty Acids (SFA) 45.7 (4.5) 43.2 (5.6) -2.50 .15

% Monounsaturated

Fatty Acids (MUFA) 19.4 (3.9) 17.2 (1.8) -2.16 .03

% Polyunsaturated

Fatty Acids (PUFA) 30.5 (7.1) 32.2 (6.5) 1.68 .47

% Omega 3 Fatty Acids (n3) 4.45 (1.9) 4.06 (1.7) -.394 .50

% Omega 6 Fatty Acids (n6) 26.0 (5.3) 28.1 (4.9) 2.08 .24

% Omega 7 Fatty Acids (n7) 5.06 (3.3) 2.01 (.46) -3.05 < .01

% Omega 9 Fatty Acids (n9) 14.4 (1.4) 15.2 (1.9) .882 .11

% Plasmologen-linked

Fatty Acids (dm) 4.44 (2.1) 7.42 (1.0) 2.98 < .01

Note. All values are represented as mole percentage of fatty acid in the erythrocyte membrane. Participant pre pistachio (PE) treatment means are shown first, followed by post pistachio (PE) treatment means. This is followed by change, which was calculated by subtracting the pre pistachio treatment means from the post pistachio treatment means. P values were calculated using a paired samples t test. n=20

Table 19

Pre and Post NP Intervention Percent Fatty Acid of Erythrocyte Membrane

Participant Mean Participant Mean

Erythrocyte Membrane PRE-NP (SD) POST-NP (SD) Change P value

% Saturated Fatty Acids (SFA) 45.3 (4.9) 44.1 (6.2) -1.21 .43

% Monounsaturated

Fatty Acids (MUFA) 19.0 (3.4) 17.4 (2.1) -1.59 .07

% Polyunsaturated

Fatty Acids (PUFA) 30.7 (6.1) 31.1 (7.0) .341 .86

% Omega 3 Fatty Acids (n3) 4.55 (1.9) 4.10 (2.1) -.457 .42

% Omega 6 Fatty Acids (n6) 26.1 (4.8) 27.0 (5.4) .803 .57

% Omega 7 Fatty Acids (n7) 4.50 (.3.2) 2.23 (.84) -2.27 < .01

% Omega 9 Fatty Acids (n9) 14.5 (1.9) 14.5 (1.9) .682 .15

% Plasmologen-linked

Fatty Acids (dm) 4.97 (2.1) 7.42 (1.3) .937 < .01

The Present Study Strengths, Limitations and Feasibility

Of the studies done on pistachio nut consumption in the healthy population, none have been done on healthy women, and none have employed the crossover design. By limiting the present study to women, results have an increased external validity when applied to women, though external validity is limited to women and results cannot be extrapolated to men. Employing a cross over design eliminated the between-participants variation. The crossover design also helps eliminate confounding variables such as seasonality or effect of the academic schedule on results. A cross-over design is not valid if there is carryover between treatments; however our 15-week washout period was more than enough time to remove carryover (Ginsberg et al., 1998).

This study relied on self-reported data from participants. Since participants were free-living basic measures to monitor compliance were taken. Study personnel collected empty bags each week from participants on the pistachio treatment. All participants completed three sets of three-day 24-hour food records which were checked for treatment compliance. Participants filled out an unusual diet diary if they consumed excluded on their treatment. The free-living design strengthened the external validity of the study, but also means participants were not visually monitored during consumption to assure full compliance.

The treatment periods, and washout period, for this study coincided with the school academic schedule. By working with the academic calendar participants were able to schedule data collection visits more easily, and the dropout rate was kept relatively low

(23%). Future studies should continue to take into account participant work or academic schedules to ensure ease of study completion.

CHAPTER 6

CONCLUSION

It was expected that pistachio consumption would not significantly impact body weight or composition, and this was confirmed in our study. Pistachio nut consumption did not have a significant impact on body composition when the pistachio and control groups were compared. However, both groups did have a mean body fat percentage and fat mass gain throughout the 10 week treatment period, which was also the 10 week academic quarter. Further studies could be done on weight management during a quarter or semester period. Studies could also be done on in a non-student or mixed student and non-student population to remove the possible quarter or semester effects.

The significant change in serum lipid levels expected in this study did not occur.

However, trends toward improvement in serum lipid levels for T-C, LDL-C, and HDL-

C/T-C was observed after participants consumed the PE diet. Additionally, a significant change was not seen in erythrocyte incorporation of fatty acids. One reason a large change may not have been seen is that our study participants had generally healthy serum lipid levels to begin with. A greater change has been seen in participants with higher baseline serum lipid levels (Sheridan et al., 2007; Sari et al., 2009). A lack of significant change in this already healthy population is a good indicator that consuming pistachio nuts regularly can contribute to the maintenance of healthy blood lipid levels, which contributes to a reduced risk of CVD. The current study population consisted of individuals who worked or went to school on the CPP campus. Further studies could be done on large scale healthy populations, with greater diversity in education and lifestyle, as the current study participants were all enrolled in college or college educated. Study

76 data on participant major was not collected, however the lab where measurements were completed was in a building where many nutrition and food science classes take place.

This proximity and nature of the study could have contributed to a higher level of nutrition and food science students in the study, which could result in a higher level of students interested in health. Future studies may want to collect data on participants’ interest in personal health.

NP. Both the PE and NP treatments decreased T-C/HDL-C and LDL-C/HDL-C, although the decrease was greater following PE treatment. The current studies non-significant but trend of improvement has similar POST-PE serum lipid levels to other post pistachio nut intervention serum lipid levels seen in healthy studies with healthy participants (Sari et al., 2010). There was zero-effect on body composition following the PE and NP diets.

These effects support the rationale that a diet high in pistachio nuts does not negatively impact weight or body composition. There was zero-effect on erythrocyte incorporation of fatty acids, and a non-significant decrease in %MUFA following both treatments, despite pistachio nuts being particularly high in MUFA. Future studies should be done on the digestion and metabolism of the dietary fats in pistachio nuts and the role it plays in the incorporation of fatty acids into the erythrocyte membrane.

Pistachio nuts can be consumed as up to 20% of daily energy without any ill effects. A free-living healthy female can consume pistachios in this quantity without a

77 concern for negative effects on body composition, serum lipids, or erythrocyte fatty acid composition.

REFERENCES

Anderson, D. A., Shapiro, J. R., & Lundgren, J. D. (2003). The freshman year of college

as a critical period for weight gain: An initial evaluation. Eating Behaviors, 4(4),

363-367.

Appel, L. J., Sacks, F. M., Carey, V. J., Obarzanek, E., Swain, J. F., Miller, E. R., . . .

Group, O. C. R. (2005). Effects of protein, monounsaturated fat, and carbohydrate

intake on blood pressure and serum lipids: Results of the OmniHeart randomized

trial. JAMA, 294(19), 2455-2464.

Baer, D. J., Gebauer, S. K., & Novotny, J. A. (2012). Measured energy value of

pistachios in the human diet. British Journal of Nutrition, 107(1), 120-125.

Barceló, F., Perona, J. S., Prades, J., Funari, S. S., Gomez-Gracia, E., Conde, M., . . .

Ruiz-Gutiérrez, V. (2009). Mediterranean-style diet effect on the structural

properties of the erythrocyte cell membrane of hypertensive patients: The

Prevencion con Dieta Mediterranea Study. Hypertension, 54(5), 1143-1150.

Berglund, L., Lefevre, M., Ginsberg, H. N., Kris-Etherton, P. M., Elmer, P. J., Stewart, P.

W., . . . Investigators, D. (2007). Comparison of monounsaturated fat with

carbohydrates as a replacement for saturated fat in subjects with a high metabolic

risk profile: Studies in the fasting and postprandial states. The American Journal

of Clinical Nutrition, 86(6), 1611-1620.

Berry, J. D., Dyer, A., Cai, X., Garside, D. B., Ning, H., Thomas, A., . . . Lloyd-Jones, D.

M. (2012). Lifetime risks of cardiovascular disease. New England Journal of

Medicine, 366(4), 321-329.

Bes-Rastrollo, M., Sabaté, J., Gómez-Gracia, E., Alonso, A., Martínez, J. A., & Martínez-

González, M. A. (2007). Nut consumption and weight gain in a Mediterranean

cohort: The SUN study. Obesity (Silver Spring), 15(1), 107-116.

Burns-Whitmore, B., Haddad, E., Sabaté, J., & Rajaram, S. (2014). Effects of

supplementing n-3 fatty acid enriched eggs and walnuts on cardiovascular disease

risk markers in healthy free-living lacto-ovo-vegetarians: A randomized,

crossover, free-living intervention study. Nutrition Journal, 13, 29.

Carter, S. J., Roberts, M. B., Salter, J., & Eaton, C. B. (2010). Relationship between

Mediterranean Diet Score and atherothrombotic risk: Findings from the Third

National Health and Nutrition Examination Survey (NHANES III), 1988-1994.

Atherosclerosis, 210(2), 630-636.

Casas-Agustench, P., Bulló, M., Ros, E., Basora, J., Salas-Salvadó, J., & investigators,

N.-P. (2011). Cross-sectional association of nut intake with adiposity in a

Mediterranean population. Nutrition, Metabolism & Cardiovascular Diseases,

21(7), 518-525.

Casas-Agustench, P., López-Uriarte, P., Bulló, M., Ros, E., Cabré-Vila, J. J., & Salas-

Salvadó, J. (2011). Effects of one serving of mixed nuts on serum lipids, insulin

resistance and inflammatory markers in patients with the metabolic syndrome.

Nutrition, Metabolism & Cardiovascular Diseases, 21(2), 126-135.

Chisholm, A., Mann, J., Skeaff, M., Frampton, C., Sutherland, W., Duncan, A., &

Tiszavari, S. (1998). A diet rich in walnuts favourably influences plasma fatty

acid profile in moderately hyperlipidaemic subjects. European Journal of Clinical

Nutrition, 52(1), 12-16.

Chisholm, A., Mc Auley, K., Mann, J., Williams, S., & Skeaff, M. (2005). Cholesterol

lowering effects of nuts compared with a Canola oil enriched cereal of similar fat

composition. Nutrition, Metabolism & Cardiovascular Diseases, 15(4), 284-292.

Cordain, L., Eaton, S. B., Sebastian, A., Mann, N., Lindeberg, S., Watkins, B. A., . . .

Brand-Miller, J. (2005). Origins and evolution of the Western diet: Health

implications for the 21st century. The American Journal of Clinical Nutrition,

81(2), 341-354.

Damasceno, N. R., Sala-Vila, A., Cofán, M., Pérez-Heras, A. M., Fitó, M., Ruiz-

Gutiérrez, V., . . . Ros, E. (2013). Mediterranean diet supplemented with nuts

reduces waist circumference and shifts lipoprotein subfractions to a less

atherogenic pattern in subjects at high cardiovascular risk. Atherosclerosis,

230(2), 347-353.

Dantas, A. P., Jiménez-Altayó, F., & Vila, E. (2012). Vascular aging: Facts and factors.

Frontiers in Physiology, 3, 325.

Djuric, Z., Ren, J., Blythe, J., VanLoon, G., & Sen, A. (2009). A Mediterranean dietary

intervention in healthy American women changes plasma carotenoids and fatty

acids in distinct clusters. Nutrition Research, 29(3), 156-163.

Dreher, M. L. (2012). Pistachio nuts: Composition and potential health benefits. Nutrition

Research, 70(4), 234-240.

Dudina, A., Cooney, M. T., Bacquer, D. D., Backer, G. D., Ducimetière, P., Jousilahti, P.,

. . . investigators, S. (2011). Relationships between body mass index,

cardiovascular mortality, and risk factors: A report from the SCORE

investigators. European Journal of Cardiovascular Prevention Rehabilitation,

18(5), 731-742.

Edwards, K., Kwaw, I., Matud, J., & Kurtz, I. (1999). Effect of pistachio nuts on serum

lipid levels in patients with moderate hypercholesterolemia. Journal of the

American College of Nutrition, 18(3), 229-232.

Estruch, R., Martínez-González, M. A., Corella, D., Salas-Salvadó, J., Ruiz-Gutiérrez, V.,

Covas, M. I., . . . Investigators, P. S. (2006). Effects of a Mediterranean-style diet

on cardiovascular risk factors: A randomized trial. Annals of Internal Medicine,

145(1), 1-11.

Estruch, R., Ros, E., Salas-Salvadó, J., Covas, M. I., Corella, D., Arós, F., . . .

Investigators, P. S. (2013). Primary prevention of cardiovascular disease with a

Mediterranean diet. New England Journal of Medicine, 368(14), 1279-1290.

Expert Panel on Detection, E. a., and Treatment of High Blood Cholesterol in Adults.

(2001). Executive Summary of The Third Report of The National Cholesterol

Education Program (NCEP) Expert Panel on Detection, Evaluation, And

Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III).

JAMA, 285(19), 2486-2497.

Fernández-Montero, A., Bes-Rastrollo, M., Barrio-López, M., de la Fuente-Arrillaga, C.,

Salas-Salvadó, J., Moreno-Galarraga, L.., . . . Investigators, P. S. (2014). Nut

consumption and 5-year all-cause mortality in a Mediterranean cohort: The SUN

Project, Nutrition.

Francis, H., & Stevenson, R. (2013). The longer-term impacts of Western diet on human

cognition and the brain. Appetite, 63, 119-128

Gebauer, S. K., West, S. G., Kay, C. D., Alaupovic, P., Bagshaw, D., & Kris-Etherton, P.

M. (2008). Effects of pistachios on cardiovascular disease risk factors and

potential mechanisms of action: A dose-response study. The American Journal of

Clinical Nutrition, 88(3), 651-659.

Ginsberg, H. N., Kris-Etherton, P., Dennis, B., Elmer, P. J., Ershow, A., Lefevre, M., . . .

Anderson, N. (1998). Effects of reducing dietary saturated fatty acids on plasma

lipids and lipoproteins in healthy subjects: The DELTA Study, protocol 1.

Arteriosclerosis, Thrombosis, and Vascular Biology, 18(3), 441-449.

Grosso, G., Pajak, A., Mistretta, A., Marventano, S., Raciti, T., Buscemi, S., . . . Galvano,

F. (2014). Protective role of the Mediterranean diet on several cardiovascular risk

factors: Evidence from Sicily, southern Italy. Nutrition, Metabolism &

Cardiovascular Diseases, 24(4), 370-377.

Gulati, S., Misra, A., Pandey, R. M., Bhatt, S. P., & Saluja, S. (2014). Effects of pistachio

nuts on body composition, metabolic, inflammatory and oxidative stress

parameters in Asian Indians with metabolic syndrome: A 24-wk, randomized

control trial. Nutrition, 30(2), 192-197.

Hodson, L., Skeaff, C. M., & Chisholm, W. A. (2001). The effect of replacing dietary

saturated fat with polyunsaturated or monounsaturated fat on plasma lipids in

free-living young adults. European Journal of Clinical Nutrition, 55(10), 908-

915.

Jenkins, D. J., Kendall, C. W., Marchie, A., Parker, T. L., Connelly, P. W., Qian, W., . . .

Spiller, G. A. (2002). Dose response of almonds on coronary heart disease risk

factors: Blood lipids, oxidized low-density lipoproteins, lipoprotein(a),

homocysteine, and pulmonary nitric oxide: A randomized, controlled, crossover

trial. Circulation, 106(11), 1327-1332.

Jurado, D., Burgos-Garrido, E., Diaz, F. J., Martínez-Ortega, J. M., & Gurpegui, M.

(2012). Adherence to the Mediterranean dietary pattern and personality in patients

attending a primary health center. Journal of the Academy of Nutrition and

Dietetics, 112(6), 887-891.

Katan, M. B., Deslypere, J. P., van Birgelen, A. P., Penders, M., & Zegwaard, M. (1997).

Kinetics of the incorporation of dietary fatty acids into serum cholesteryl esters,

erythrocyte membranes, and adipose tissue: An 18-month controlled study.

Journal of Lipid Research, 38(10), 2012-2022.

Kocyigit, A., Koylu, A. A., & Keles, H. (2006). Effects of pistachio nuts consumption on

plasma lipid profile and oxidative status in healthy volunteers. Nutrition,

Metabolism & Cardiovascular Diseases, 16(3), 202-209.

Kris-Etherton, P. M., Pearson, T. A., Wan, Y., Hargrove, R. L., Moriarty, K., Fishell, V.,

& Etherton, T. D. (1999). High-monounsaturated fatty acid diets lower both

plasma cholesterol and triacylglycerol concentrations. American Journal of

Clinical Nutrition, 70(6), 1009-1015.

Li, Z., Song, R., Nguyen, C., Zerlin, A., Karp, H., Naowamondhol, K., . . . Heber, D.

(2010). Pistachio nuts reduce triglycerides and body weight by comparison to

refined carbohydrate snack in obese subjects on a 12-week weight loss program.

Journal of the American College of Nutrition, 29(3), 198-203.

Martínez-González, M. A., & Bes-Rastrollo, M. (2011). Nut consumption, weight gain

and obesity: Epidemiological evidence. Nutrition, Metabolism & Cardiovascular

Diseases, 21 Suppl 1, S40-45.

Mathieu, P., Poirier, P., Pibarot, P., Lemieux, I., & Després, J. P. (2009). Visceral

obesity: The link among inflammation, hypertension, and cardiovascular disease.

Hypertension, 53(4), 577-584.

McGuire, S. (2011). U.S. Department of Agriculture and U.S. Department of Health and

Human Services, Dietary Guidelines for Americans, 2010. 7th Edition,

Washington, DC: U.S. Government Printing Office, January 2011. Advances in

Nutrition, 2(3), 293-294.

McKay, D. L., Chen, C. Y., Yeum, K. J., Matthan, N. R., Lichtenstein, A. H., &

Blumberg, J. B. (2010). Chronic and acute effects of walnuts on antioxidant

capacity and nutritional status in humans: A randomized, cross-over pilot study.

Nutrition Journal, 9, 21.

Miller, M., Stone, N. J., Ballantyne, C., Bittner, V., Criqui, M. H., Ginsberg, H. N., . . .

Disease, C. o. t. K. i. C. (2011). Triglycerides and cardiovascular disease: A

scientific statement from the American Heart Association. Circulation, 123(20),

2292-2333.

Mosca, L., Benjamin, E. J., Berra, K., Bezanson, J. L., Dolor, R. J., Lloyd-Jones, D. M., .

. . Wenger, N. K. (2011). Effectiveness-based guidelines for the prevention of

cardiovascular disease in women--2011 update: A guideline from the American

Heart Association. Circulation, 123(11), 1243-1262.

Mozaffarian, D., Micha, R., & Wallace, S. (2010). Effects on coronary heart disease of

increasing polyunsaturated fat in place of saturated fat: A systematic review and

meta-analysis of randomized controlled trials. PLoS Med, 7(3), e1000252.

Murphy, S.L., Xu, J.Q., Kochanek, K.D. (2013) Deaths: Final data for 2010. National

vital statistics reports; 61(4). Hyattsville, MD: National Center for Health

Statistics.

Novotny, J. A., Gebauer, S. K., & Baer, D. J. (2012). Discrepancy between the Atwater

factor predicted and empirically measured energy values of almonds in human

diets. American Journal of Clinical Nutrition, 96(2), 296-301.

O'Neil, C. E., Keast, D. R., Nicklas, T. A., & Fulgoni, V. L. (2012). Out-of-hand nut

consumption is associated with improved nutrient intake and health risk markers

in US children and adults: National Health and Nutrition Examination Survey

1999-2004. Nutrition Research, 32(3), 185-194.

Obarzanek, E., Sacks, F. M., Vollmer, W. M., Bray, G. A., Miller, E. R., Lin, P. H., . . .

Group, D. R. (2001). Effects on blood lipids of a blood pressure-lowering diet:

The Dietary Approaches to Stop Hypertension (DASH) Trial. American Journal

of Clinical Nutrition, 74(1), 80-89.

Pieterse, Z., Jerling, J. C., Oosthuizen, W., Kruger, H. S., Hanekom, S. M., Smuts, C. M.,

& Schutte, A. E. (2005). Substitution of high monounsaturated fatty acid avocado

for mixed dietary fats during an energy-restricted diet: Effects on weight loss,

serum lipids, fibrinogen, and vascular function. Nutrition, 21(1), 67-75

Rajaram, S., Haddad, E. H., Mejia, A., & Sabaté, J. (2009). Walnuts and fatty fish

influence different serum lipid fractions in normal to mildly hyperlipidemic

individuals: A randomized controlled study. American Journal of Clinical

Nutrition, 89(5), 1657S-1663S.

Ros, E., Núñez, I., Pérez-Heras, A., Serra, M., Gilabert, R., Casals, E., & Deulofeu, R.

(2004). A walnut diet improves endothelial function in hypercholesterolemic

subjects: A randomized crossover trial. Circulation, 109(13), 1609-1614.

Sabaté, J., Fraser, G. E., Burke, K., Knutsen, S. F., Bennett, H., & Lindsted, K. D. (1993).

Effects of walnuts on serum lipid levels and blood pressure in normal men. New

England Journal of Medicine, 328(9), 603-607.

Sari, I., Baltaci, Y., Bagci, C., Davutoglu, V., Erel, O., Celik, H., . . . Aksoy, M. (2010).

Effect of pistachio diet on lipid parameters, endothelial function, inflammation,

and oxidative status: A prospective study. Nutrition, 26(4), 399-404.

Schwingshackl, L., & Hoffmann, G. (2012). Monounsaturated fatty acids and risk of

cardiovascular disease: Synopsis of the evidence available from systematic

reviews and meta-analyses. Nutrients, 4(12), 1989-2007.

Sheridan, M. J., Cooper, J. N., Erario, M., & Cheifetz, C. E. (2007). Pistachio nut

consumption and serum lipid levels. Journal of the American College of

Nutrition, 26(2), 141-148.

Simopoulos, A. P. (2003). Essential fatty acids in health and chronic diseases. Forum of

Nutrition, 56, 67-70.

Stone, N. J., Nicolosi, R. J., Kris-Etherton, P., Ernst, N. D., Krauss, R. M., & Winston,

M. (1996). AHA conference proceedings. Summary of the scientific conference

on the efficacy of hypocholesterolemic dietary interventions. American Heart

Association. Circulation, 94(12), 3388-3391.

Traoret, C. J., Lokko, P., Cruz, A. C., Oliveira, C. G., Costa, N. M., Bressan, J., . . .

Mattes, R. D. (2008). Peanut digestion and energy balance. International Journal

of Obesity (London), 32(2), 322-328.

Vijayakrishnan, R., Kalyatanda, G., Srinivasan, I., & Abraham, G. M. (2013).

Compliance with the Adult Treatment Panel III guidelines for hyperlipidemia in a

resident-run ambulatory clinic: A retrospective data analysis. Journal of Clinical

Lipidology, 7(1), 43-47.

Wien, M. A., Sabaté, J. M., Iklé, D. N., Cole, S. E., & Kandeel, F. R. (2003). Almonds vs

complex carbohydrates in a weight reduction program. International Journal of

Obesity and Related Metabolic Disorders, 27(11), 1365-1372.

Zambón, D., Sabaté, J., Muñoz, S., Campero, B., Casals, E., Merlos, M., . . . Ros, E.

(2000). Substituting walnuts for monounsaturated fat improves the serum lipid

profile of hypercholesterolemic men and women. A randomized crossover trial.

Annals of Internal Medicine, 132(7), 538-546.

APPENDIX A

IRB APPROVAL

APPENDIX B

SAMPLE RECRUITMENT E-MAIL

Cal Poly Pomona Department of Human Nutrition and Food Science IRB Protocol #12-139

The Cal Polys Pistachio Study Are you:

* A non-pregnant female?

* Between 18 and 40 years of age?

* Healthy? (i.e. no chronic diseases)

Do you:

* Not consume alcohol regularly?

* Like pistachios? If so, you may qualify to participate in a study examining the effects of pistachio on body composition, inflammatory and lipid levels! The study will be completed on both the Cal Poly Pomona and SLO campuses during Fall and Spring Quarters.

* Be willing to eat a bag per day of pistachios for 12 weeks during Fall or Spring quarter.

* Be willing not to eat pistachios for another 12 weeks during either Fall or Spring quarter.

* Fast for 8 hours before a blood draw and have your blood drawn 4 times by the Student Health Center.

* Have your body composition analyzed 4 times.

* Complete a 3-day food record 7 times, and Food Satiety Questionnaires 7 times.

YOU WILL RECEIVE A $10 FARM STORE CARD AS COMPENSATION

If you feel that you qualify, and are willing to participate, please send an e-mail to either: Amy Towne (Graduate Student) at [email protected] or Dr. Bonny Burns-Whitmore at [email protected]

APPENDIX C

INFORMED CONSENT FORM

California State Polytechnic University, Pomona Informed Consent Form for Research Involving Human Subjects

You are being invited to participate in a research study, which the Cal Poly Pomona Institutional Review Board (IRB) has reviewed and approved for conduct by the investigators named here. This form is designed to provide you - as a human subject - with information about this study. The Investigator or his/her representative will describe this study to you and answer any of your questions. You are entitled to an Experimental Research Subject’s Bill of Rights and a copy of this form. If you have any questions about your rights as a subject, complaints about the informed consent process of this research study, or experience an adverse event (something goes wrong), please contact the Compliance Office within Cal Poly Pomona’s Office of Research at (909) 869-4215. More information is available at the IRB website, www.csupomona.edu/research/irb.

Effects of Pistachio Consumption on Body Composition, Bone Density, Satiety, Inflammatory Markers and Fatty Acid Analysis IRB Protocol # 12-139 (CPP)

Researchers:

Dr. Laura Hall (Principal Investigator), Assistant Professor, Food Science and Nutrition Department, California State Polytechnic University, San Luis Obispo, [email protected]; (805) 756-2958 Dr. Bonny Burns-Whitmore (Co-Investigator), RD, Professor, Department of Human Nutrition and Food Science, California Polytechnic University, Pomona; [email protected]; (909) 869-3793

Researchers’ Statement:

We are asking you to participate in a research study. The purpose of this consent form is to give you the information you will need to help you decide if you would like to participate. The study will begin on the September 17, 2012, and will continue for a total of 39 weeks. Please read the form carefully. If there is anything that is unclear, please do not hesitate to obtain clarification from the researchers. When we have answered all your questions, please decide if you would like to be included in the study. This process is called “informed consent”. You will be provided with a copy of this form for your records. Cal Poly San Luis Obispo has received approval from their Institutional Review Board on 09-11-12. The data from this study will be compiled from both campuses and analyzed. Your name will not appear on any of the data sheets.

PURPOSE OF THE STUDY The overall purpose of this study is to examine the effects of pistachio consumption on body composition, bone density, markers of bone turnover, erythrocyte membrane incorporation of fatty acids, lipid status, inflammatory markers, dietary quality/micronutrient quality and satiety using a cross-over study with two 12-week treatments (pistachio added (20% of kcals) and a no- pistachio control diet), separated by 15-week washout period. We will recruit 30 female students from Cal Poly, San Luis Obispo (CP-SLO) and female 30 students from Cal Poly, Pomona (CP- P) facilitating a collaborative project and using the expertise of both Dr. Laura Hall (CP-SLO)

APPENDIX C (Continued)

INFORMED CONSENT FORM and Dr. Bonny Burns-Whitmore (CP-P). We hope to show the health benefits of pistachios as part of an overall healthy lifestyle. Cal Poly Pomona will be looking at the effects of eating a diet rich in pistachios on body composition, red blood cell membrane changes, blood lipids, a marker of inflammation (CRP), and satiety.

STUDY PROCEDURES—Diagram of the study. You will be assigned to either eating pistachios for 12 weeks in Fall quarter or not eating them. However, if you are not eating them in Fall quarter, you will eat them in Spring quarter.

Lead-in Treatment #1 Rest period Treatment #2 1 week 12 weeks 15 weeks 12 weeks *^# *^# *^# *^# *^# *^# *^# *^# (3) ‡ (9) ‡ (9) ‡

Lead-in Pistachios-weekly pick up of supply Control-Pistachio -free diet Blood draw * Randomized diet records/satiety questions ‡ DXA-bone density (CPSLO) ^ Tanita-body composition #

Treatment #1 will either be the pistachio or pistachio free treatment. Treatment two will be the opposite of Treatment #1.

1. After signing the consent form, you will fill out a brief health-screening and qualification questionnaire. a. Examples of questions will be: -Are you currently suffering from or have you ever been diagnosed with a chronic disease (heart attack, cancer, diabetes, renal failure, asthma)? -How many pistachios do you eat on a weekly basis? b. For important safety reasons, you will be asked to answer each and every question on the health-screening questionnaire prior to qualification for the study; especially the nut-allergy and medication log. We are asking you for this, because we are concerned about your safety.

2. You will be educated/instructed about the background diet and exercise plan that you must maintain for the duration of the study. The background diet/habitual diet is your normal diet, but free from pistachios or pistachio-based products. This instruction will require you to meet with the study personnel, a couple of days before the study starts, for a total of 25 total study visits.

APPENDIX C (Continued)

INFORMED CONSENT FORM

3. This study is composed of two distinct experimental treatments (12 weeks each) with a 15- week washout (rest period) diet in-between the treatment diets: a. The pistachio-free diet, in which you must maintain just the background diet. The pistachio-added diet, in which you must consume 20% of your calories of pistachio per day in addition to the background diet. We will ask that you meet with the study personnel every week during the pistachio treatment to pick up your weekly allotment of nuts. There will be a 15-week washout (rest period) period during Winter quarter. This includes the holiday break. We want you to continue to eat your regular diet during this time. The study will resume in Spring quarter. NOTE: The order in which you complete these two treatments will be randomly determined at the beginning of the study. The two experimental treatments will be separated by a 15-week period in which only the background diet is consumed.

4. Before you begin the first experimental phase, you will complete a one-week “run-in” period in which the background diet will be consumed and we will ask you to complete 3 diet records.

5. At the beginning and end of each distinct period (treatments), we will ask for you to fast for 8 hours before your blood draw. Your blood will be drawn and assessed for total cholesterol, HDL, LDL cholesterol, CRP, and a regular blood lab screening (glucose, calcium, potassium, iron, sodium, etc…) by a trained phlebotomist at the Cal Poly Pomona Student Health Center (via appointment). One additional tube will be taken for red blood cell membrane analysis. The blood draw will take approximately 20 minutes and the Health Center will draw about 3 tubes; which is about 3 tablespoons.

6. Your body composition will be measured at these same beginning and end of each distinct treatment using a Tanita brand bioimpedance analyzer. The body composition measurements will require you to remove your shoes and socks, and stand on a platform while the machine analyzes body composition. The analysis will take approximately 10 minutes. This measurement will be taken twice.

7. Your blood pressure will be measured at these same intervals using an Omicron blood pressure cuff. The blood pressure measurement will require you to allow us to measure your blood pressure with a wrist cuff that will expand and then deflate, revealing your blood pressure and heart rate. You will be asked to sit down and have your blood pressure measured using a blood pressure cuff around your left wrist. This will take less than 1 minute. This measurement will be taken twice.

8. At 7 random points during each of the diet period you will be asked to fill out a three-day food record and activity recall log. Each food and activity recall log will take approximately 1 hour to complete.

9. Your waist circumference will be measured using a flexible measuring tape between the middle of the bottom rib (close to your belly button) and iliac crest (top of the hip bone) and recorded to the nearest 0.1 cm. Hip circumference will be measured using a flexible measuring tape around the largest part of the hips (i.e. buttocks) and recorded to the nearest 0.1 cm. This measurement will be taken twice. It will take no longer than 5 minutes to do both measurements.

APPENDIX C (Continued)

INFORMED CONSENT FORM

10. Your height will be measured using a stadiometer and recorded to the nearest 0.1 cm. This will take no longer than 5 minutes to do and will only be done once at the beginning of the study.

11. You will be asked to rate your hunger using a validated rating scale (1-10) and questionnaire next to each snack or meal on the 7 required 3-day food records.

INCLUSION CRITERIA

You are qualified to participate in this study if you: -are a female. -are between the ages of 18 and 40. -are not averse to consuming pistachios on a regular basis (at least 12 weeks) -have not taken any medication for any chronic disease (heart, diabetes, cancer) for 12 weeks -have not taken any steroid or hormone medication in the last 8 weeks -are free of any other chronic diseases. -do not consume excess amounts of nuts -do not consume alcohol on a regular basis -are not currently on a diet plan. -are not pregnant or plan to get pregnant

Do you meet all the criteria listed above? ______YES ______NO

EXCLUSION CRITERIA

You are NOT qualified to participate in this study if you: -are a child, teenager, woman, or male younger than 18 or older than 40. -eat large quantities of pistachios on a regular basis. -are taking any steroid or hormone medication.(other than birth control pills) -are pregnant or become pregnant -are diagnosed with any chronic diseases. -are currently on a diet plan. -have a known allergy to nuts -consume excess amounts of nuts -consume alcohol on a regular basis -have a pacemaker or metal pins or plates in the body

Do you meet any of the criteria listed above? ______YES ______NO

This study will be conducted from the California Polytechnic University, Pomona campus. You will be required to come to campus each week (place to be determined as per your schedule) to pick up your allotment of pistachios, and will be required to visit the Student Health Center for a blood draw 4 times. You will be asked to fast for 8 hours before the blood draw.

APPENDIX C (Continued)

INFORMED CONSENT FORM

RISKS, STRESS, OR DISCOMFORT

It is possible that regularly consuming pistachios will result in mild gastrointestinal discomfort. If you experience these, or other symptoms, in connection with this study, please do not hesitate to get in touch with Dr. Burns-Whitmore at (909) 869-3793, or your physician to discuss options.

If more severe or intolerable symptoms are experienced, please contact Dr. Burns-Whitmore at (909) 869-3793 or (805) 231-0864-cell, the Student Health Center, the emergency room at the nearest hospital, 911 or your physician and immediately discontinue eating the pistachios.

Symptoms of an allergic reaction or intolerance include scratchy throat, difficulty breathing and/or swallowing, skin rash, blurred vision, persistent diarrhea, vomiting, and excessive gastrointestinal discomfort.

It is possible that you may experience some discomfort from the blood draw. Sometimes blood draws may cause a vein to collapse, which means that another vein may need to be used. A collapsed vein leaves a very large bruise, which should begin to dissipate after a couple of days. If the bruise doesn’t go away or seems to spread, or you experience pain, please contact the Health Center or your physician, and Dr. Burns-Whitmore. You may possibly experience anemia from the blood draw, so if you feel dizzy or light-headed or weak after the blood draw, please contact the Health Center or your physician, and Dr. Burns-Whitmore.

Dr. Burns-Whitmore can be contacted at (909) 869-3793 or [email protected].

If your blood values are found to be not within the normal range, we will ask you to have the values re-checked at the Student Health Center.

It is possible that you might experience a weight gain, however, a number of published scientific studies show that adding 20% of your required calories in the form of nuts will not cause significant weight gain.

BENEFITS OF THIS STUDY

You will receive free pistachios for 12 weeks and compensation in the form of a gift card to the Farm Store at California Polytechnic University, Pomona worth $10.00 at the end of the study. You will also find out the results of your body composition as well as other measurements.

PARTICIPATION IN THE STUDY Your participation in this study is voluntary, and declining to participate will involve no penalty or loss of benefits to which you are otherwise entitled, and you may discontinue participation at any time without penalty. You will receive compensation pro-rated in the form of a gift card to the Cal Poly Farm Store for your participation. For example if you only complete one treatment, you will receive a $5.00 gift card instead of $10.You will also receive a certificate of thanks from the study personnel at the end of the study or if you cannot complete the study.

APPENDIX C (Continued)

INFORMED CONSENT FORM

OTHER INFORMATION

Any information provided by you will be confidential. In the event that the investigators learn that any participant intends to harm herself or others, this intent must be reported to the authorities. University staff sometimes reviews studies to ensure that they are being done safely, legally, and ethically. The reviewers will protect your privacy, and study records will not be used to harm any person or put him at legal risk.

COMPENSATION FOR INJURY

There will be no compensation for lost wages, lost time, allergic reactions, adverse reactions, or pain. Payment for care resulting from adverse reactions is the sole responsibility of the participant and they should consider having medical insurance to pay for such care.

PRINTED NAME OF STAFF/PERSON OBTAINING CONSENT SIGNATURE DATE

SUBJECT’S STATEMENT

This study has been explained to me. I volunteer to take part in this research. I have had a chance to ask questions. If I have any questions later about the study, I can ask one of the researchers listed above. If I have questions about my rights as a research subject or a research-related injury, I can call the Compliance Office within Cal Poly Pomona’s Office of Research at (909) 869- 4215. I will receive a copy of this consent form.

PRINTED NAME OF SUBJECT SIGNATURE DATE

EXPERIMENTAL RESEARCH SUBJECT’S BILL OF RIGHTS

California law, under Health & Safety Code Section 24172, requires that any person asked to take part as a subject in research involving a medical experiment, or any person asked to consent to such participation on behalf of another, is entitled to receive the following list of rights written in a language in which the person is fluent. This list includes the right to: 1. Be informed of the nature and purpose of the experiment. 2. Be given an explanation of the procedures to be followed in the medical experiment, and any drug or device to be utilized. 3. Be given a description of any attendant discomforts and risks reasonably to be expected from the experiment. 4. Be given an explanation of any benefits to the subject reasonably to be expected from the experiment, if applicable. 5. Be given a disclosure of any appropriate alternative procedures, drugs or devices that might be advantageous to the subject, and their relative risks and benefits.

APPENDIX C (Continued)

INFORMED CONSENT FORM

6. Be informed of the avenues of medical treatment, if any, available to the subject after the experiment if complications should arise. 7. Be given an opportunity to ask any questions concerning the experiment or the procedures involved. 8. Be instructed that consent to participate in the medical experiment may be withdrawn at any time and the subject may discontinue participation in the medical experiment without prejudice. 9. Be given a copy of the signed and dated written consent form. 10. Be given the opportunity to decide to consent or not to consent to a medical experiment without the intervention of any element of force, fraud, deceit, duress, coercion, or undue influence on the subject’s decision.

APPENDIX D

INITIAL SCREENING QUESTIONAIRE

APPENDIX D (Continued)

INITIAL SCREENING QUESTIONAIRE

100

APPENDIX D (Continued)

INITIAL SCREENING QUESTIONAIRE

101

APPENDIX E

INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE

102

APPENDIX E (Continued)

INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE

103

APPENDIX E (Continued)

INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE

104

APPENDIX F

SAMPLE 24-HOUR DIET RECORD

105

APPENDIX F (Continued)

SAMPLE 24-HOUR DIET RECORD

106

APPENDIX F (Continued)

SAMPLE 24-HOUR DIET RECORD

107

APPENDIX G

SAMPLE UNUSUAL DIET DIARY

108

APPENDIX G (Continued)

SAMPLE UNUSUAL DIET DIARY

109

APPENDIX G (Continued)

SAMPLE UNUSUAL DIET DIARY

110

APPENDIX H

JOURNAL ARTICLE

The effects of pistachio nuts on body composition and serum lipids of healthy, free-living females

Amy Towne, Bonny Burns-Whitmore, David Edens and Golandam Khayef

ABSTRACT Risk of cardiovascular disease is lower in those with healthy serum lipid levels and healthy body composition. A diet high in nuts has been shown to improve or maintain body composition and serum lipid levels. The present study sought to determine the effects of regular pistachio nut consumption in young, healthy, free-living, female participants. This study utilized a cross-over design with two 10-week treatment periods, pistachio enriched diet (PE) in which 20% of daily calories were provided by pistachios and no-pistachio control diet (NP), separated by a 15-week washout period. At the beginning and end of each treatment period, following a 12-hour overnight fast, participants’ blood lipids, waist circumference, hip circumference, body weight and body composition using bioelectrical impedance analysis measurements were taken. There were no significant differences in body composition or serum lipid between PE and NP. Baseline and post-treatment serum lipid and body composition values were within ideal ranges for lowered cardiovascular disease risk. Improvement was seen in the mean lowering of total cholesterol, LDL cholesterol and in total cholesterol to HDL cholesterol and in LDL cholesterol to HDL cholesterol (ratios), following the PE treatment. A diet rich in pistachio nuts does not contribute to changes in body composition and may potentially improve serum lipids in young, free-living, healthy, females.

INTRODUCTION According to the Centers for Disease Control’s (CDC) National Vital Statistics Report, cardiovascular diseases are the leading cause of death in the United States(1). The percentage of death from cardiovascular diseases (CVD) in those with an optimal risk factor profile is 4.7% in men and 6.4% in women, whereas the percentage death from cardiovascular diseases in those with two or more major risk factors is 29.6% in men and 20.5% in women (2). Current smoking, diabetes, hypercholesterolemia, total cholesterol level of 240 mg per deciliter, hypertension, and systolic blood pressure of 160 mm Hg or diastolic blood pressure of 100mm Hg, along with obesity and an increase in visceral fat have been identified as risk factors for the development of CVD(2-3). There is strong evidence that incorporating foods that are high in monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have a beneficial effect on blood lipid levels (4- 5). Whole foods high in MUFAs and PUFAs while low in saturated fatty acids (SFA), such as tree nuts and avocado, have been targeted in dietary interventions (6-7). Different nuts contain different percentages of MUFA, PUFA, and SFA. Therefore, individual nuts are studies to understand their specific effects (6, 8-9). The purpose of this study is to examine the effects of regular pistachio consumption on blood lipid profiles, erythrocyte incorporation of fatty acids, and body composition on free-living healthy females, age 18- 40. To measure effects on blood lipid profiles, collected and analyzed serum lipid levels

111

APPENDIX H (Continued)

JOURNAL ARTICLE and erythrocyte incorporation of fatty acids at baseline and post-intervention for each treatment period.

SUBJECTS AND METHODS Subject criteria In order to be eligible for this study, participants had to be students or staff at CPP, biologically female, between ages 18 and 40, at a body mass index (BMI) between 18.5 and 25, and free of chronic diseases. Additionally, participants were asked if that liked pistachio nuts. Persons were not eligible for this study, if they were not a student or staff at CPP, if they were not biologically female, if they were less than 18 years old or older than 40 years old, or had a BMI less than 18.5 or greater than 25. Interested persons were not eligible if they were allergic to pistachios or other nuts or if they ate an excessive amount of nuts. Additionally, if the interested person had a disease or chronic condition that required treatment that may affect metabolism, dietary intake, or physical activity levels, they were not eligible. The use of medications that affect fat malabsorption, laxative use, liver disease, kidney disease, thyroid disease, diabetes, hypertension, dyslipidemia, pregnancy, intent to become pregnant, excessive use of alcohol, chronic disease, cancer, pacemakers or metal pins, plates in the body, or unwillingness to follow the study protocol which involves not consuming other nuts, fish or flaxseed oil or supplements, excluded interested persons from participating in the study. Lastly, interested persons who were elite athletes were also excluded from participating in this study. Mean participant characteristics are reported in tables 1 and 2.

Table 1 Mean Participant Baseline, Pre-pistachio Treatment, Anthropometric Characteristics

Standard Measure Mean Deviation Minimum Maximum

Age (years) 22.8 4.45 18.0 33.0 Height (cm) 164 7.29 151 181 Weight (kg) 60.3 7.89 44.8 73.5 Body Mass Index (BMI) 22.3 2.20 18.2 27.3 Body Fat Percentage (BFP) 26.1 5.49 14.5 36.3 Fat Mass (kg) 16.1 5.29 6.50 26.7 Fat Free Mass (kg) 44.2 3.23 38.3 49.5 Body Water (kg) 32.4 2.36 28.1 36.3 Waist Circumference (cm) 28.6 1.89 25.2 32.0 Hip Circumference (cm) 39.0 2.02 35.5 43.3

Note. All baseline anthropometric values are reported from pre-pistachio treatment, obtained during week 0 prior to the start of 10-week pistachio treatment. n=23.

112

APPENDIX H (Continued)

JOURNAL ARTICLE

Table 2 Mean Participant Baseline, Pre-pistachio Treatment, Serum Lipid Profile

Standard Measure Mean Deviation Minimum Maximum

LDL-C 79.0 26.1 20.0 140 HDL-C 60.0 13.7 23.0 83.0 T-C 154 32.4 79.0 223 TAG 74.3 34.7 37.0 161 T-C/HDL (ratio) 2.65 0.57 1.34 3.96 LDL-C/HDL-C (ratio) 1.38 .503 .240 2.64

Note. All baseline serum lipid values are reported from pre-pistachio treatment, obtained during week 0 prior to the start of 10-week pistachio treatment. n=23.

Experimental design This study employed a cross-over study design. In this design, participants participated in each of the following interventions 1)No-pistachio diet(NP), in which participants abstained from eating pistachio or other nuts and 2) Pistachio-enriched diet (PE), in which participants consumed pistachios nuts daily, in a provided amount which equaled 20% of their daily calorie intake. Participants were asked not to consume other nuts. Each dietary treatment would by 10 weeks long, which was determined as sufficiently long for the stabilization on serum lipids (10). All participants completed a 15-week washout period in between treatment periods, during which participants consumed their regular diets. Fasting blood draws and body composition analyses were completed at baseline and post-intervention of each treatment period.

STATISTICAL ANALYSIS Raw data were entered into Microsoft Office Excel 2007 spreadsheets. Body composition, serum lipids, and erythrocyte fatty acid composition taken before the pistachio treatment were entered in as variable X one, the post pistachio treatment values were entered in as variable X two, the body composition and blood values taken before the no pistachio control treatment were entered in as variable X three, the values taken post no pistachio control treatment were entered in as variable X four. Pre and post treatment differences were calculated by subtracting variable X one from variable X two (XDIFF12), and variable X three from variable X four (XDIFF34). Differences were calculated for body composition, serum lipids, and erythrocyte fatty acid composition variables. The data set created in Excel was imported into SPSS (version 22), for statistical analysis. Based on recommendations from Tabachnick & Fidell (2007) assumptions of normality were checked (11). Based on recommendations from Morgan (2007) a

113

APPENDIX H (Continued)

JOURNAL ARTICLE comparison of means for the variables expressing the difference in treatments, “XDIFF12” and “XDIFF34”, was determined using a paired samples t test (12).

RESULTS Thirty-one women, recruited from CPP enrolled in the study. One participant had a negative reaction to having blood drawn and dropped out during the initial week of the study; the participant was replaced by the end of the first week, resulting in 30 enrolled participants. Of the 30 women recruited, seven women dropped out during the course of the study, leaving 23 participants (n=23). The average participant baseline, pre-pistachio treatment, age was 22 ± 4 years with average BMI of 22 ± 2. The average participant total cholesterol (T-C) was 154 ± 32 with an LDL of 79 ± 26. The average participant erythrocyte %MUFA was 19 ± 4, %PUFA was 30 ± 7. Characteristics reported as pre pistachio treatment means ± standard deviation. Results of a paired samples t test indicated that participants’ body weight did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = 1.20, p = .24, d = .24. The difference is not statistically significant. Participant body weight did not significantly change pre and post pistachio treatment, t (22) = 1.51, p = .14, d = .32. Participants’ lean mass did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = .89, p = .381, d = .19. The difference is not statistically significant. Participant lean mass did not significantly change pre and post pistachio treatment, t (22) = -1.59, p = .13, d = -.33. Participants’ waist to hip ratio did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = 1.20, p = .25, d = .25. The difference is not statistically significant. Participant waist to hip ratio did not significantly change pre and post pistachio treatment t (22) = .89, p = .38, d .19. Results of a paired samples t test indicated that participant total cholesterol did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = -.40, p = .690, d = -.08. The difference is not statistically significant. Participant total cholesterol did not significantly change pre and post pistachio treatment t (22) = -.56, p = .60, d -.11. Participants’ LDL-C did not significantly change during regular pistachio consumption, when compared to the no pistachio control, t (22) = -.75, p = .463, d = -.16. The difference is not statistically significant. Participant LDL-C did not significantly change pre and post pistachio treatment t (22) = -.70, p = .49, d -.15. Participants’ HDL-C did not significantly change during regular pistachio consumption, when compared to no pistachio control, t (22) = -.03, p = .980, d = -.01. The difference is not statistically significant. Participant HDL-C did not significantly change pre and post pistachio treatment t (22) = -.28, p = .78, d -.06. Participants’ blood triacylglycerols did not significantly change during regular pistachio consumption, compared to no pistachio control, t (22) = 1.51, p = .145, d = .32. The difference is not statistically significant. Participant Triacylglycerol did not significantly change pre and post pistachio treatment t (22) = .60, p = .55, d .13.

114

APPENDIX H (Continued)

JOURNAL ARTICLE

DISCUSSION

Body Composition There were no significant results when comparing body composition changes during the PE diet to body composition changes during the NP diet. These results are similar to other nut consumption studies where participant diets were isocaloric and activity level was maintained (8-9). Serum Lipids Following the PE diet there were non-significant favorable mean changes in LDL- C t (22) = -.70, p = .42, d = -.15 in T-C, t (22) = -.55, p = .59, d = -.11 in T-C/HDL-C, t (22) = -.72, p = .48, d = -.15 and also in LDL-C/HDL-C, t (22) = -.90, p = .38, d = -.19. Figure 4 show mean changes of the pistachio treatment on LDL-C, T-C, T-C/HDL-C and LDL-C/HDL-C. There were no significant serum lipid changes when comparing changes in serum lipids during the PE and NP treatments. Other studies have shown changes in serum lipids during pistachio treatments (13-15). These studies were done in non-healthy participants, which include hypercholesterolemic participants, those with elevated LDL-C or those with metabolic syndrome (13-15). Other pistachio consumption studies done in healthy participants have shown a reduction in serum lipid levels, in contrast to what was seen in the current study (14, 16- 17). Kocyigit (2006) completed a study on healthy individuals consuming 20 percent of their daily calories from pistachio nuts over three weeks and found a significant decrease in T-C, T-C/HDL-C, and LDL-C/HDL-C. The Kocyigit (2006) study varied from the present study as participants in the pistachio group consumed reduced portions of fatty foods such as meat, and the amount of visible fats consumed was decreased to accommodate the percentage of energy from pistachio nuts. Therefore, the Kocyigit (2006) study results are more of a reflection of purposefully replacing saturated fats with the fats from pistachio nuts, as opposed to the current study which did not instruct participants to reduce their saturated fat intake. Sari (2010) found a significant reduction in LDL-C, T-C and TAG, however the baseline values for those in this study were at the high end of the ideal range: LDL-C mean 124.5, T-C mean 190.7, and TAG mean 112.1, and lowered down to the following means LDL-C mean 95, T-C mean 149.4 and TAG mean 91.1. The post intervention means in the Sari (2010) study were similar to, or still above the current studies baseline and post intervention means. Baer (2012) found a significant reduction in LDL-C when three varying pistachio doses were consumed for eighteen consecutive days. The study also found that dietary fat in pistachio nuts is poorly metabolized (17). The metabolisable energy (ME) value of pistachio nuts was found to be less than the Atwater general factors, which is how energy content of food is primarily determined. Additionally, there was a notable inter-individual variability in ME values within the eighteen study participants. The current study did not examine the energy value of nuts and, therefore, we do not know how efficiently the examined participants metabolized the fats from the pistachio nuts.

115

APPENDIX H (Continued)

JOURNAL ARTICLE

Overall, the regular consumption of pistachio nuts had a trend toward improvement in the serum lipids LDL-C, T-C, T-C/HDL-C and LDL-C/HDL-C. The trend toward improvement was seen comparing PRE-PE treatment means to POST-PE treatment means. The decrease in LDL-C and T-C seen POST-PE was not seen POST- NP. Both the PE and NP treatments decreased T-C/HDL-C and LDL-C/HDL-C, although the decrease was greater following PE treatment. The current studies non-significant but trend of improvement has similar POST-PE serum lipid levels to other post pistachio nut intervention serum lipid levels seen in healthy studies with healthy participants (Sari et al., 2010). There was zero-effect on body composition following the PE and NP diets. These effects support the rationale that a diet high in pistachio nuts does not negatively impact weight or body composition. There was zero-effect on erythrocyte incorporation of fatty acids, and a non-significant decrease in %MUFA following both treatments, despite pistachio nuts being particularly high in MUFA. Future studies should be done on the digestion and metabolism of the dietary fats in pistachio nuts and the role it plays in the incorporation of fatty acids into the erythrocyte membrane. Pistachio nuts can be consumed as up to 20% of daily energy without any ill effects. A free-living healthy female can consume pistachios in this quantity without a concern for negative effects on body composition, serum lipids, or erythrocyte fatty acid composition.

116

APPENDIX H (Continued)

JOURNAL ARTICLE

REFERENCES

1. Heron M. Deaths: leading causes for 2010. Natl Vital Stat Rep. 2013;62(6):1-96. 2. Berry JD, Dyer A, Cai X, et al. Lifetime risks of cardiovascular disease. N Engl J Med. 2012;366(4):321-329. 3. Dudina A, Cooney MT, Bacquer DD, et al. Relationships between body mass index, cardiovascular mortality, and risk factors: a report from the SCORE investigators. Eur J Cardiovasc Prev Rehabil. 2011;18(5):731-742. 4. Appel LJ, Sacks FM, Carey VJ, et al. Effects of protein, monounsaturated fat, and carbohydrate intake on blood pressure and serum lipids: results of the OmniHeart randomized trial. JAMA. 2005;294(19):2455-2464. 5. Berglund L, Lefevre M, Ginsberg HN, et al. Comparison of monounsaturated fat with carbohydrates as a replacement for saturated fat in subjects with a high metabolic risk profile: studies in the fasting and postprandial states. Am J Clin Nutr. 2007;86(6):1611-1620. 6. Wien MA, Sabaté JM, Iklé DN, Cole SE, Kandeel FR. Almonds vs complex carbohydrates in a weight reduction program. Int J Obes Relat Metab Disord. 2003;27(11):1365-1372. 7. Pieterse Z, Jerling JC, Oosthuizen W, et al. Substitution of high monounsaturated fatty acid avocado for mixed dietary fats during an energy-restricted diet: effects on weight loss, serum lipids, fibrinogen, and vascular function. Nutrition. 2005;21(1):67-75. 8. Chisholm A, Mann J, Skeaff M, et al. A diet rich in walnuts favourably influences plasma fatty acid profile in moderately hyperlipidaemic subjects. Eur J Clin Nutr. 1998;52(1):12-16. 9. Zambón D, Sabaté J, Muñoz S, et al. Substituting walnuts for monounsaturated fat improves the serum lipid profile of hypercholesterolemic men and women. A randomized crossover trial. Ann Intern Med. 2000;132(7):538-546. 10. Ginsberg HN, Kris-Etherton P, Dennis B, et al. Effects of reducing dietary saturated fatty acids on plasma lipids and lipoproteins in healthy subjects: the DELTA Study, protocol 1. Arterioscler Thromb Vasc Biol. 1998;18(3):441-449. 11. Tabachnick BG, Fidell LS. Using multivariate statistics. 5th ed. ed. Boston, Mass. ; London: Pearson/Allyn & Bacon; 2007. 12. Morgan GA. SPSS for introductory statistics : use and interpretation. 3rd ed. ed. Mahwah, N.J. ; London: Lawrence Erlbaum; 2007. 13. Edwards K, Kwaw I, Matud J, Kurtz I. Effect of pistachio nuts on serum lipid levels in patients with moderate hypercholesterolemia. J Am Coll Nutr. 1999;18(3):229-232. 14. Kocyigit A, Koylu AA, Keles H. Effects of pistachio nuts consumption on plasma lipid profile and oxidative status in healthy volunteers. Nutr Metab Cardiovasc Dis. 2006;16(3):202-209. 15. Sheridan MJ, Cooper JN, Erario M, Cheifetz CE. Pistachio nut consumption and serum lipid levels. J Am Coll Nutr. 2007;26(2):141-148.

117

16. Sari I, Baltaci Y, Bagci C, et al. Effect of pistachio diet on lipid parameters, endothelial function, inflammation, and oxidative status: a prospective study. Nutrition. 2010;26(4):399-404. 17. Baer DJ, Gebauer SK, Novotny JA. Measured energy value of pistachios in the human diet. Br J Nutr. 2012;107(1):120-125.

118