JOURNAL OF MEDICINAL FOOD J Med Food 17 (5) 2014, 612–617 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.0034
Drinking Orange Juice Increases Total Antioxidant Status and Decreases Lipid Peroxidation in Adults Shahrzad Foroudi,1 Andrew S. Potter,1 Alexis Stamatikos,1 Bhimanagouda S. Patil,2 and Farzad Deyhim1–3 1
Department of Human Sciences, Texas A&M University–Kingsville, Kingsville, Texas, USA. Department of Horticultural Sciences, Vegetable and Fruit Improvement Center, Texas A&M University, College Station, Texas, USA. 3 Citrus Center, Texas A&M University–Kingsville, Weslaco, Texas, USA.
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ABSTRACT Cardiovascular disease (CVD) is the leading cause of death in the world and is the primary cause of mortality among Americans. One of the many reasons for the pathogenesis of CVD is attributed to eating diets high in saturated fat and refined carbohydrates and low in fruits and vegetables. Epidemiological evidence has supported a strong association between eating diets rich in fruits and vegetables and cardiovascular health. An experiment was conducted utilizing 24 adults with hypercholesterolemia and hypertriglyceridemia to evaluate the impact of drinking 20 fl oz of freshly squeezed orange juice daily for 90 days on blood pressure, lipid panels, plasma antioxidant capacity, metabolic hormones, lipid peroxidation, and inflammatory markers. Except for addition of drinking orange juice, subjects did not modify their eating habits. The findings suggested that drinking orange juice does not affect (P > .1) blood pressure, lipid panels, metabolic hormones, body fat percentage, or inflammatory markers. However, total plasma antioxidant capacity was significantly increased (P < .05) and lipid peroxidation was significantly decreased (P < .05) after orange juice consumption. Drinking orange juice may protect the cardiovascular system by increasing total plasma antioxidant status and by lowering lipid peroxidation independent of other cardiovascular risk markers evaluated in this study. KEY WORDS: antioxidant status cardiovascular disease lipid peroxidation orange juice
C-reactive protein, insulin resistance, and dyslipidemia,10 which are also considered to be risk factors for CVD.11–13 Increased consumption of fruits and vegetables and other dietary modifications have been shown to reduce the obesity rate and lower the risk of CVD. In a recent study, an inverse association between eating fruits and vegetables with obesity and CVD was observed.14 It has been reported that eating five servings of fruits decreased the risk of CVD by 7%.15 In another meta-analysis of cohort studies, increased intake of fruits and vegetables from less than three servings per day to more than five servings per day reduced the incidence of CVD by 17%.16 Although there are a number of studies that concluded diets high in fruits and vegetables and low in saturated fat lower the risk of developing obesity and CVD,14,16,17 others have reported that daily consumption of five or more servings of fruits and vegetables, set by the USDA guidelines, is rarely met.18,19 In an effort to improve fruit consumption, we believe drinking 20 fl oz of freshly squeezed orange juice (2½ cups, *591.5 mL) is approximately equal to half of the USDA Daily Food Plan recommendation of 4½ cups of fruits and vegetables for a reference 2000 calorie diet.20 Potential benefits of increased consumption of fruits and vegetables are improved antioxidant status,21–23 increased
INTRODUCTION
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ccording to the American Heart Association, cardiovascular disease (CVD) is the leading cause of death in the United States.1 Approximately 864,000 Americans die each year from CVD, accounting for 35% of the total deaths in the United States.2 Worldwide, it is estimated that by year 2030, 23.6 million people will die from CVD.3 Diets high in fat are modifiable risk factor in the development of CVD and stroke.1 It is believed that eating diets high in saturated fat cause 30% of CVDs.1,2 Eating diets high in saturated fat and cholesterol contribute to an increase in total plasma cholesterol and low-density lipoprotein cholesterol levels, which are known to be risk factors for CVD.4–6 Another risk factor that affects CVD occurrence is the metabolic syndrome, which contributes to the increase in CVD mortality.7–9 In addition, obesity is thought to be a risk factor that initiates a cascade of events, which leads to systemic inflammation and the development of increased circulating Manuscript received 4 February 2013. Revision accepted 5 November 2013. Address correspondence to: Farzad Deyhim, PhD, RD, LD, Department of Human Sciences, Texas A&M University-Kingsville, MSC 168, Kingsville, TX 78363, USA, E-mail: [email protected]
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circulating micronutrient concentrations,22,24 reduced proinflammatory markers and improved anti-inflammatory markers,15,25–29 reduced CVD biomarkers,22,29 reduced CVD incidence,15,30–33 and a lowered CVD-related mortality rate.34–35 Two studies have observed inverse associations between the intake of citrus fruits and incidence CVD.36,37 In animal studies, we reported that drinking orange juice or eating orange pulp increased plasma antioxidant capacity, improved liver antioxidant enzymes, and suppressed lipid peroxidation.38,39 Data from other studies have suggested that oxidative stress increases the atherogenicity of low-density lipoprotein, promotes atherogenesis, and increases the risk of CVD.40,41 Fruits and vegetables have been shown to be rich sources of phenolic compounds with high antioxidant capacity, which may have anti-atherogenic properties thereby decreasing the risk of vascular disease42,43 and protecting against CVD.44 A previous study has shown potential benefits of fruit and vegetable juice concentrates on cardiovascular prevention.22 The aim of the present study was to evaluate the potential role of daily consumption of 20 fl oz of freshly squeezed orange juice (2½ cups) on lowering cardiovascular risk markers in adults with elevated cholesterol and triglycerides. The 20 fl oz of freshly squeezed orange juice follows the recommendation made by the USDA Daily Food Plan.20 METHODS Demographics of the study population The research study was approved by the TAMUK Institutional Review Board (IRB) before initiation of the study. Participants in this study were 24 adults, 12 males and 12 females, with elevated plasma cholesterol and triglyceride levels. Participants’ age ranged from 45 to 55 years. Participants were asked not to take multivitamins and minerals for the duration of the study. Subjects did not use tobacco nor were they taking prescription medications. Subjects were asked not to change their lifestyle other than drinking the orange juice. Participation was totally voluntary. Participants agreed to drink the orange juice daily and signed a consent form in agreement to the rules and regulations of the study. Orange juice was delivered to the research participants daily. According to the USDA National Nutrient Database for Standard Reference, 20 fl oz orange juice provides 547.46 mL water, 279 kcal, 52.08 g sugar, 1.2 g fiber, 4.34 g protein, 1.24 g lipid, 68 mg Ca, 105 mg P, 1240 mg K, 6 mg Na, 186 lg folate, and 310 mg vitamin C.45 Each subject was asked to drink 20 fl oz of juice daily for the 3-month duration (90 days) of the study as a morning snack. Written informed consent was obtained from all participants. Weight and height were measured both pretest and post-test to calculate body mass index (BMI) status. Fasting blood samples were collected and plasma was harvested by a licensed nurse at the start of the study and after 90 days when the study was concluded. Blood was centrifuged at 1500 g for 15 min to obtain the plasma. Lipid panels, including triglyceride, cho-
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lesterol, high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), apolipoprotein A (Apo A), apolipoprotein B (Apo B), and lipoprotein [Lp (a)], were analyzed using the Modular Analytics D 2400 system and Cobas Integra 800 of Roche Diagnostics Corp. (Indianapolis, IN, USA). Outcome measures Blood pressure collection. Three blood pressure readings from the left upper arm were taken and the values were averaged using the OMRON Model #HEM 711 at the beginning and end of the study. Blood pressure was taken from each subject and repeated if (1) an error occurred with the reading, (2) subjects seemed anxious or nervous, or (3) blood pressure measurement was above the normal range (120 mm Hg/80 mm Hg). Bioelectrical impedance analysis. Bioelectrical impedance analysis (BIA) technique was used to estimate the percentage of body fat using Quantum II (RJL Systems, 2006, Clinton Twp., MI, USA). The BIA test was performed while each subject was lying supine with their arms and legs spread open. After the electrode site was cleaned with isopropyl alcohol, electrode patches with self-adhesive conducting gel were attached to the dorsal surface of the right foot and right hand. The electrodes introduced an alternating current (50 kHz) at the base of the toes and fingers with the Quantum II measuring the voltage changes. Body fat percentages as determined by BIA were measured at the beginning and end of the study. Inflammatory markers and hormones. Plasma interleukin-1a was analyzed as a proinflammatory marker (R&D System, Minneapolis, MN, USA). Plasma C-reactive protein was analyzed using a C-reactive protein ELISA kit as an index for inflammation (Life Diagnostics, Westchester, PA, USA). Plasma insulin was evaluated to assess insulin resistance (Linco Research, Inc., St. Charles, MI, USA). Leptin was analyzed as an adiposity and satiety hormone (Linco Research, Inc.). Insulin-like growth factor-I (IGF-1) was measured to indicate metabolic and hormonal activity (Linco Research, Inc.). Homocysteine was assessed as a CVD risk marker (MyBioSource, LLC., San Diego, CA, USA). Total antioxidant status and malondialdehyde production. After the plasma was obtained, an aliquot was refrigerated for total antioxidant status using a commercially available kit (Calbiochem, San Diego, CA, USA) as a quantitative measure of circulating antioxidant status and for plasma malondialdehyde using a kit from Northwest Life Science (Vancouver, WA, USA) as an indicator of lipid peroxidation. Statistics The study was a pretest/post-test and a paired t-test was performed to determine the effects of drinking 20 fl oz fresh orange juice daily as an independent variable on variables of
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interest at baseline and 90 days after the initiation of the study, as outlined by Steel and Terrie.46
Variables
RESULTS Drinking 20 fl oz of orange juice daily for 3 months did not (P > .1) alter the subject’s weight, body fat percentage, or BMI (Table 1). Drinking orange juice had no impact on blood pressure or fasting chemistry, including lipid panels like triglycerides, cholesterol, HDL, LDL, and VLDL, or insulin, IGF-1, and leptin. Plasma Apo A, Apo B, Lp (a), and homocysteine were not (P > .1) affected by drinking orange juice (Table 1). Plasma inflammatory markers, interleukin1a and C-reactive protein, were not (P > .1) affected by drinking orange juice (Table 1). However, plasma antioxidant status was significantly (P < .05) increased, while plasma malondialdehyde concentration was significantly (P < .05) decreased after drinking orange juice (Table 1). Comparing males to females, male participants weighed more than female participants (Table 2). The lipid panel, blood pressure, insulin, IGF-1, and plasma antioxidant capacity were Table 1. Effects of Drinking Orange Juice on Anthropometrics, Blood Pressure, Lipid Panel, Hormones, Inflammatory Markers, and Antioxidant Status
Anthropometric Age (years) Body weight (kg) BMI Fat% Blood pressure Systolic (mm Hg) Diastolic (mm Hg) Lipid panel TAG (mg/dL) Cholesterol (mg/dL) HDL (mg/dL) VLDL (mg/dL) LDL (mg/dL) Apo A (mg/dL) Apo B (mg/dL) Lp (a) (mg/dL) Homocysteine (nm/mL) Hormones IGF-1 (ng/mL) Insulin (lU/mL) Leptin (ng/mL) Inflammatory markers Interleukin-1a (pg/mL) C-reactive protein (mg/L) Antioxidant status Total antioxidant capacity (mM) Malondialdehyde (lM)
Table 2. Gender Differences on Anthropometrics, Blood Pressure, and Lipid Panels
Pretest
Post-test
50 – 2 91 – 5 31 – 1 29 – 2
50 – 2 92 – 5 31 – 1 33 – 2
128 – 3 76 – 2
128 – 3 75 – 2
144 – 11 221 – 8 50 – 3 29 – 2 143 – 6 144 – 6 103 – 3 12.4 – 2 10 – 0.73
158 – 11 236 – 8 51 – 3 32 – 3 150 – 6 151 – 6 109 – 4 13.9 – 2 11 – 0.73
2.25 – 0.39 19 – 3 22 – 2.8
1.55 – 0.2 20 – 3 21 – 2.8
0.808 – 0.13 6.99 – 0.70
0.956 – 0.20 8.82 – 0.70
1.25 – 0.09b 40 – 5a
1.51 – 0.09a 20 – 5b
ab Means – SEMs with unlike superscript are significantly (P £ .05) different from each other. HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; Apo, apolipoprotein; Lp (a), lipoprotein; BMI, body mass index.
Age (years) Pre Post Body weight (Kg) Pre Post BMI Pre Post Fat (%) Pre Post Systolic pressure (mm Hg) Pre Post Diastolic pressure (mm Hg) Pre Post TAG (mg/dL) Pre Post Cholesterol (mg/dL) Pre Post HDL (mg/dL) Pre Post VLDL (mg/dL) Pre Post LDL (mg/dL) Pre Post
Female
Male
50 – 5 50 – 5
50 – 5 50 – 5
78 – 4 79 – 4
104 – 5 106 – 5
31 – 2 31 – 2
31 – 2 31 – 2
41 – 2a 45 – 2a
18 – 2b 20 – 2b
125 – 11 125 – 11
131 – 11 132 – 11
77 – 8 76 – 7
76 – 8 74 – 8
132 – 15 154 – 15
156 – 15 162 – 15
228 – 12 245 – 12
214 – 11 225 – 11
55 – 3a 58 – 3a
45 – 3b 43 – 3b
25 – 3 31 – 3
31 – 3 32 – 3
146 – 8 160 – 8
140 – 8 140 – 8
ab Means – SEMs with unlike superscript are significantly (P £ .05) different from each other.
not (P > .1) different between male and female participants (Tables 2 and 3). Despite the higher (P < .05) percentage of body fat, leptin, and C-reactive protein in females, plasma concentrations of homocysteine and malondialdehyde were significantly (P < .05) higher in males (Table 3). DISCUSSION Studies pertaining to the dietary intake of fruits and vegetables on CVD risk present several difficulties. These include controlling individual nutrient intake, lifestyle factors, and varying eating habits between subjects along with the inability to monitor eating patterns between subjects. In the present study, we have examined the effect of drinking 20 fl oz of orange juice on CVD risk markers independent of the overall eating pattern by individual subjects. This method of evaluation on CVD risk markers provides a more realistic measure of exposure since eating habits most likely differ drastically among subjects.
DRINKING ORANGE JUICE AND CARDIOVASCULAR HEALTH Table 3. Gender Differences on Cardiovascular, Endocrine, Inflammatory Marker, and Antioxidant Status Variables
Female
Apo A (mg/dL) Pre 151 – 8 Post 154 – 8 Apo B (mg/dL) Pre 102 – 5 Post 108 – 5 Lp (a) (mg/dL) Pre 13.6 – 3 Post 16.8 – 3 Homocysteine (nm/mL) Pre 8.8 – 0.7c Post 10.0 – 0.7bc IGF-1 (ng/mL) Pre 2.43 – 0.4 Post 1.83 – 0.4 Insulin (lU/mL) Pre 18.7 – 3 Post 22.8 – 3 Leptin (ng/mL) Pre 34 – 3a Post 33 – 3a Interleukin-1a (pg/mL) Pre 0.874 – 0.47 Post 1.098 – 0.47 C-reactive protein (mg/L) Pre 8.18 – 0.73a Post 10.74 – 0.72a Total antioxidant capacity (mM) Pre 1.19 – 0.10c Post 1.42 – 0.10b Malondialdehyde (lM) Pre 34 – 3b Post 10 – 3d
Male 148 – 8 139 – 8 104 – 5 109 – 5 11.2 – 3 10.9 – 3 11.2 – 0.7ab 12.6 – 0.7a 2.06 – 0.4 1.27 – 0.4 19.4 – 3 18.1 – 3 10.03 – 2b 8.85 – 2b 0.742 – 0.31 0.813 – 0.31 5.80 – 0.73b 6.90 – 0.73b 1.30 – 0.12b 1.72 – 0.12a 46 – 3a 28 – 3bc
abcd Means – SEMs with unlike superscript are significantly (P £ .05) different from each other.
In the present study, the lack of effects of drinking freshly squeezed orange juice on cardiovascular markers may well be attributed to the fact that no change in the eating pattern was requested. Subjects were asked to not modify their eating habits with the exception of consuming 20 fl oz of orange juice as a morning snack. Despite no changes in anthropometric values, lipid panels, insulin, leptin, and inflammatory markers, orange juice contributed to a significant 25% increase in antioxidant status, as determined by total antioxidant status, as well as a marked 50% reduction in lipid peroxidation, as determined by pretest and post-test malondialdehyde concentrations. The nutrient content of orange juice most likely contributed to the effect observed in improving antioxidant status and suppressing lipid peroxidation and is consistent with our earlier human and animal studies.23,38 This study disagrees with those who reported that drinking 20 fl oz of blood orange juice has no effect on the total antioxidant capacity and malondialdehyde concentrations.47 The discrepancy may be related to the duration of the study. In our study,
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subjects drank freshly squeezed orange juice for 3 months, whereas in the study conducted by Riso et al., subjects drank blood orange juice for 3 weeks.47 However, this study agrees with those who reported that drinking *6.7 fl oz of orange juice with one apple and a pear daily for 26 days increased the plasma total antioxidant capacity.48 Moreover, decreased lipid peroxidation evident from drinking orange juice is associated with increased antioxidant status independent of inflammatory markers, hormones, or cholesterol and triglyceride levels. Similar to our previous carrot juice study, drinking orange juice did not affect plasma insulin, leptin, IGF-1, interleukin1a, homocysteine, and C-reactive protein, which may have been because laboratory values were all within normal ranges like the beginning of the study.23 We hypothesize that high mean cholesterol and triglyceride concentrations observed in this study are actually attributed to the eating habits and lifestyle factors. In study conducted by Alvarez-Parrilla,48 subjects consumed one apple, one pear, and *6.7 fl oz of orange juice for 26 days, and their total cholesterol, HDL cholesterol, and LDL cholesterol increased significantly, but the increase in concentrations did not exceed the normal range set by the health authorities.48 Discrepancy in results reported in this study compared with an earlier report may be related to the duration of the study, age of the subjects, combination of eating three fruits containing fiber, and overall eating habits. As expected, there were physiological differences that existed between male and female participants. As anticipated and reported in other studies, men weighed more and had a lower body fat percentage than their female counterparts.23,49,50 In contrast, female participants had higher HDL and higher leptin concentrations. The higher plasma leptin and HDL levels in females agree with an earlier report and are attributed to sex hormones, as observed in another study.23 However, elevated plasma homocysteine and malondialdehyde observed in men suggest that they are at a greater risk for developing CVD than women participants and it may partly be related to men experiencing higher levels of oxidative stress than women. In conclusion, drinking 20 fl oz of freshly squeezed orange juice daily significantly increased antioxidant status and reduced malondialdehyde, which is a marker for lipid peroxidation, without affecting plasma cholesterol and triglyceride status.
ACKNOWLEDGMENTS This project is based upon work supported by the USDACSREES nos. 2008-34402-19195 and 2010-34402-20875 ‘‘Designing Foods for Health’’ through the Vegetable & Fruit Improvement Center. AUTHOR DISCLOSURE STATEMENT The authors declare that they have no competing interests. REFERENCES 1. American Heart Association: Heart Disease and Stroke Statistics. 2005; http://my.clevelandclinic.org/Documents/heart/1105390918119 HDSStats2005Update.pdf (accessed December 2012).
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Drinking Orange Juice Increases Total Antioxidant Status and Decreases Lipid Peroxidation in Adults Shahrzad Foroudi,1 Andrew S. Potter,1 Alexis Stamatikos,1 Bhimanagouda S. Patil,2 and Farzad Deyhim1–3 1
Department of Human Sciences, Texas A&M University–Kingsville, Kingsville, Texas, USA. Department of Horticultural Sciences, Vegetable and Fruit Improvement Center, Texas A&M University, College Station, Texas, USA. 3 Citrus Center, Texas A&M University–Kingsville, Weslaco, Texas, USA.
2
ABSTRACT Cardiovascular disease (CVD) is the leading cause of death in the world and is the primary cause of mortality among Americans. One of the many reasons for the pathogenesis of CVD is attributed to eating diets high in saturated fat and refined carbohydrates and low in fruits and vegetables. Epidemiological evidence has supported a strong association between eating diets rich in fruits and vegetables and cardiovascular health. An experiment was conducted utilizing 24 adults with hypercholesterolemia and hypertriglyceridemia to evaluate the impact of drinking 20 fl oz of freshly squeezed orange juice daily for 90 days on blood pressure, lipid panels, plasma antioxidant capacity, metabolic hormones, lipid peroxidation, and inflammatory markers. Except for addition of drinking orange juice, subjects did not modify their eating habits. The findings suggested that drinking orange juice does not affect (P > .1) blood pressure, lipid panels, metabolic hormones, body fat percentage, or inflammatory markers. However, total plasma antioxidant capacity was significantly increased (P < .05) and lipid peroxidation was significantly decreased (P < .05) after orange juice consumption. Drinking orange juice may protect the cardiovascular system by increasing total plasma antioxidant status and by lowering lipid peroxidation independent of other cardiovascular risk markers evaluated in this study. KEY WORDS: antioxidant status cardiovascular disease lipid peroxidation orange juice
C-reactive protein, insulin resistance, and dyslipidemia,10 which are also considered to be risk factors for CVD.11–13 Increased consumption of fruits and vegetables and other dietary modifications have been shown to reduce the obesity rate and lower the risk of CVD. In a recent study, an inverse association between eating fruits and vegetables with obesity and CVD was observed.14 It has been reported that eating five servings of fruits decreased the risk of CVD by 7%.15 In another meta-analysis of cohort studies, increased intake of fruits and vegetables from less than three servings per day to more than five servings per day reduced the incidence of CVD by 17%.16 Although there are a number of studies that concluded diets high in fruits and vegetables and low in saturated fat lower the risk of developing obesity and CVD,14,16,17 others have reported that daily consumption of five or more servings of fruits and vegetables, set by the USDA guidelines, is rarely met.18,19 In an effort to improve fruit consumption, we believe drinking 20 fl oz of freshly squeezed orange juice (2½ cups, *591.5 mL) is approximately equal to half of the USDA Daily Food Plan recommendation of 4½ cups of fruits and vegetables for a reference 2000 calorie diet.20 Potential benefits of increased consumption of fruits and vegetables are improved antioxidant status,21–23 increased
INTRODUCTION
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ccording to the American Heart Association, cardiovascular disease (CVD) is the leading cause of death in the United States.1 Approximately 864,000 Americans die each year from CVD, accounting for 35% of the total deaths in the United States.2 Worldwide, it is estimated that by year 2030, 23.6 million people will die from CVD.3 Diets high in fat are modifiable risk factor in the development of CVD and stroke.1 It is believed that eating diets high in saturated fat cause 30% of CVDs.1,2 Eating diets high in saturated fat and cholesterol contribute to an increase in total plasma cholesterol and low-density lipoprotein cholesterol levels, which are known to be risk factors for CVD.4–6 Another risk factor that affects CVD occurrence is the metabolic syndrome, which contributes to the increase in CVD mortality.7–9 In addition, obesity is thought to be a risk factor that initiates a cascade of events, which leads to systemic inflammation and the development of increased circulating Manuscript received 4 February 2013. Revision accepted 5 November 2013. Address correspondence to: Farzad Deyhim, PhD, RD, LD, Department of Human Sciences, Texas A&M University-Kingsville, MSC 168, Kingsville, TX 78363, USA, E-mail: [email protected]
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circulating micronutrient concentrations,22,24 reduced proinflammatory markers and improved anti-inflammatory markers,15,25–29 reduced CVD biomarkers,22,29 reduced CVD incidence,15,30–33 and a lowered CVD-related mortality rate.34–35 Two studies have observed inverse associations between the intake of citrus fruits and incidence CVD.36,37 In animal studies, we reported that drinking orange juice or eating orange pulp increased plasma antioxidant capacity, improved liver antioxidant enzymes, and suppressed lipid peroxidation.38,39 Data from other studies have suggested that oxidative stress increases the atherogenicity of low-density lipoprotein, promotes atherogenesis, and increases the risk of CVD.40,41 Fruits and vegetables have been shown to be rich sources of phenolic compounds with high antioxidant capacity, which may have anti-atherogenic properties thereby decreasing the risk of vascular disease42,43 and protecting against CVD.44 A previous study has shown potential benefits of fruit and vegetable juice concentrates on cardiovascular prevention.22 The aim of the present study was to evaluate the potential role of daily consumption of 20 fl oz of freshly squeezed orange juice (2½ cups) on lowering cardiovascular risk markers in adults with elevated cholesterol and triglycerides. The 20 fl oz of freshly squeezed orange juice follows the recommendation made by the USDA Daily Food Plan.20 METHODS Demographics of the study population The research study was approved by the TAMUK Institutional Review Board (IRB) before initiation of the study. Participants in this study were 24 adults, 12 males and 12 females, with elevated plasma cholesterol and triglyceride levels. Participants’ age ranged from 45 to 55 years. Participants were asked not to take multivitamins and minerals for the duration of the study. Subjects did not use tobacco nor were they taking prescription medications. Subjects were asked not to change their lifestyle other than drinking the orange juice. Participation was totally voluntary. Participants agreed to drink the orange juice daily and signed a consent form in agreement to the rules and regulations of the study. Orange juice was delivered to the research participants daily. According to the USDA National Nutrient Database for Standard Reference, 20 fl oz orange juice provides 547.46 mL water, 279 kcal, 52.08 g sugar, 1.2 g fiber, 4.34 g protein, 1.24 g lipid, 68 mg Ca, 105 mg P, 1240 mg K, 6 mg Na, 186 lg folate, and 310 mg vitamin C.45 Each subject was asked to drink 20 fl oz of juice daily for the 3-month duration (90 days) of the study as a morning snack. Written informed consent was obtained from all participants. Weight and height were measured both pretest and post-test to calculate body mass index (BMI) status. Fasting blood samples were collected and plasma was harvested by a licensed nurse at the start of the study and after 90 days when the study was concluded. Blood was centrifuged at 1500 g for 15 min to obtain the plasma. Lipid panels, including triglyceride, cho-
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lesterol, high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), apolipoprotein A (Apo A), apolipoprotein B (Apo B), and lipoprotein [Lp (a)], were analyzed using the Modular Analytics D 2400 system and Cobas Integra 800 of Roche Diagnostics Corp. (Indianapolis, IN, USA). Outcome measures Blood pressure collection. Three blood pressure readings from the left upper arm were taken and the values were averaged using the OMRON Model #HEM 711 at the beginning and end of the study. Blood pressure was taken from each subject and repeated if (1) an error occurred with the reading, (2) subjects seemed anxious or nervous, or (3) blood pressure measurement was above the normal range (120 mm Hg/80 mm Hg). Bioelectrical impedance analysis. Bioelectrical impedance analysis (BIA) technique was used to estimate the percentage of body fat using Quantum II (RJL Systems, 2006, Clinton Twp., MI, USA). The BIA test was performed while each subject was lying supine with their arms and legs spread open. After the electrode site was cleaned with isopropyl alcohol, electrode patches with self-adhesive conducting gel were attached to the dorsal surface of the right foot and right hand. The electrodes introduced an alternating current (50 kHz) at the base of the toes and fingers with the Quantum II measuring the voltage changes. Body fat percentages as determined by BIA were measured at the beginning and end of the study. Inflammatory markers and hormones. Plasma interleukin-1a was analyzed as a proinflammatory marker (R&D System, Minneapolis, MN, USA). Plasma C-reactive protein was analyzed using a C-reactive protein ELISA kit as an index for inflammation (Life Diagnostics, Westchester, PA, USA). Plasma insulin was evaluated to assess insulin resistance (Linco Research, Inc., St. Charles, MI, USA). Leptin was analyzed as an adiposity and satiety hormone (Linco Research, Inc.). Insulin-like growth factor-I (IGF-1) was measured to indicate metabolic and hormonal activity (Linco Research, Inc.). Homocysteine was assessed as a CVD risk marker (MyBioSource, LLC., San Diego, CA, USA). Total antioxidant status and malondialdehyde production. After the plasma was obtained, an aliquot was refrigerated for total antioxidant status using a commercially available kit (Calbiochem, San Diego, CA, USA) as a quantitative measure of circulating antioxidant status and for plasma malondialdehyde using a kit from Northwest Life Science (Vancouver, WA, USA) as an indicator of lipid peroxidation. Statistics The study was a pretest/post-test and a paired t-test was performed to determine the effects of drinking 20 fl oz fresh orange juice daily as an independent variable on variables of
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interest at baseline and 90 days after the initiation of the study, as outlined by Steel and Terrie.46
Variables
RESULTS Drinking 20 fl oz of orange juice daily for 3 months did not (P > .1) alter the subject’s weight, body fat percentage, or BMI (Table 1). Drinking orange juice had no impact on blood pressure or fasting chemistry, including lipid panels like triglycerides, cholesterol, HDL, LDL, and VLDL, or insulin, IGF-1, and leptin. Plasma Apo A, Apo B, Lp (a), and homocysteine were not (P > .1) affected by drinking orange juice (Table 1). Plasma inflammatory markers, interleukin1a and C-reactive protein, were not (P > .1) affected by drinking orange juice (Table 1). However, plasma antioxidant status was significantly (P < .05) increased, while plasma malondialdehyde concentration was significantly (P < .05) decreased after drinking orange juice (Table 1). Comparing males to females, male participants weighed more than female participants (Table 2). The lipid panel, blood pressure, insulin, IGF-1, and plasma antioxidant capacity were Table 1. Effects of Drinking Orange Juice on Anthropometrics, Blood Pressure, Lipid Panel, Hormones, Inflammatory Markers, and Antioxidant Status
Anthropometric Age (years) Body weight (kg) BMI Fat% Blood pressure Systolic (mm Hg) Diastolic (mm Hg) Lipid panel TAG (mg/dL) Cholesterol (mg/dL) HDL (mg/dL) VLDL (mg/dL) LDL (mg/dL) Apo A (mg/dL) Apo B (mg/dL) Lp (a) (mg/dL) Homocysteine (nm/mL) Hormones IGF-1 (ng/mL) Insulin (lU/mL) Leptin (ng/mL) Inflammatory markers Interleukin-1a (pg/mL) C-reactive protein (mg/L) Antioxidant status Total antioxidant capacity (mM) Malondialdehyde (lM)
Table 2. Gender Differences on Anthropometrics, Blood Pressure, and Lipid Panels
Pretest
Post-test
50 – 2 91 – 5 31 – 1 29 – 2
50 – 2 92 – 5 31 – 1 33 – 2
128 – 3 76 – 2
128 – 3 75 – 2
144 – 11 221 – 8 50 – 3 29 – 2 143 – 6 144 – 6 103 – 3 12.4 – 2 10 – 0.73
158 – 11 236 – 8 51 – 3 32 – 3 150 – 6 151 – 6 109 – 4 13.9 – 2 11 – 0.73
2.25 – 0.39 19 – 3 22 – 2.8
1.55 – 0.2 20 – 3 21 – 2.8
0.808 – 0.13 6.99 – 0.70
0.956 – 0.20 8.82 – 0.70
1.25 – 0.09b 40 – 5a
1.51 – 0.09a 20 – 5b
ab Means – SEMs with unlike superscript are significantly (P £ .05) different from each other. HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; Apo, apolipoprotein; Lp (a), lipoprotein; BMI, body mass index.
Age (years) Pre Post Body weight (Kg) Pre Post BMI Pre Post Fat (%) Pre Post Systolic pressure (mm Hg) Pre Post Diastolic pressure (mm Hg) Pre Post TAG (mg/dL) Pre Post Cholesterol (mg/dL) Pre Post HDL (mg/dL) Pre Post VLDL (mg/dL) Pre Post LDL (mg/dL) Pre Post
Female
Male
50 – 5 50 – 5
50 – 5 50 – 5
78 – 4 79 – 4
104 – 5 106 – 5
31 – 2 31 – 2
31 – 2 31 – 2
41 – 2a 45 – 2a
18 – 2b 20 – 2b
125 – 11 125 – 11
131 – 11 132 – 11
77 – 8 76 – 7
76 – 8 74 – 8
132 – 15 154 – 15
156 – 15 162 – 15
228 – 12 245 – 12
214 – 11 225 – 11
55 – 3a 58 – 3a
45 – 3b 43 – 3b
25 – 3 31 – 3
31 – 3 32 – 3
146 – 8 160 – 8
140 – 8 140 – 8
ab Means – SEMs with unlike superscript are significantly (P £ .05) different from each other.
not (P > .1) different between male and female participants (Tables 2 and 3). Despite the higher (P < .05) percentage of body fat, leptin, and C-reactive protein in females, plasma concentrations of homocysteine and malondialdehyde were significantly (P < .05) higher in males (Table 3). DISCUSSION Studies pertaining to the dietary intake of fruits and vegetables on CVD risk present several difficulties. These include controlling individual nutrient intake, lifestyle factors, and varying eating habits between subjects along with the inability to monitor eating patterns between subjects. In the present study, we have examined the effect of drinking 20 fl oz of orange juice on CVD risk markers independent of the overall eating pattern by individual subjects. This method of evaluation on CVD risk markers provides a more realistic measure of exposure since eating habits most likely differ drastically among subjects.
DRINKING ORANGE JUICE AND CARDIOVASCULAR HEALTH Table 3. Gender Differences on Cardiovascular, Endocrine, Inflammatory Marker, and Antioxidant Status Variables
Female
Apo A (mg/dL) Pre 151 – 8 Post 154 – 8 Apo B (mg/dL) Pre 102 – 5 Post 108 – 5 Lp (a) (mg/dL) Pre 13.6 – 3 Post 16.8 – 3 Homocysteine (nm/mL) Pre 8.8 – 0.7c Post 10.0 – 0.7bc IGF-1 (ng/mL) Pre 2.43 – 0.4 Post 1.83 – 0.4 Insulin (lU/mL) Pre 18.7 – 3 Post 22.8 – 3 Leptin (ng/mL) Pre 34 – 3a Post 33 – 3a Interleukin-1a (pg/mL) Pre 0.874 – 0.47 Post 1.098 – 0.47 C-reactive protein (mg/L) Pre 8.18 – 0.73a Post 10.74 – 0.72a Total antioxidant capacity (mM) Pre 1.19 – 0.10c Post 1.42 – 0.10b Malondialdehyde (lM) Pre 34 – 3b Post 10 – 3d
Male 148 – 8 139 – 8 104 – 5 109 – 5 11.2 – 3 10.9 – 3 11.2 – 0.7ab 12.6 – 0.7a 2.06 – 0.4 1.27 – 0.4 19.4 – 3 18.1 – 3 10.03 – 2b 8.85 – 2b 0.742 – 0.31 0.813 – 0.31 5.80 – 0.73b 6.90 – 0.73b 1.30 – 0.12b 1.72 – 0.12a 46 – 3a 28 – 3bc
abcd Means – SEMs with unlike superscript are significantly (P £ .05) different from each other.
In the present study, the lack of effects of drinking freshly squeezed orange juice on cardiovascular markers may well be attributed to the fact that no change in the eating pattern was requested. Subjects were asked to not modify their eating habits with the exception of consuming 20 fl oz of orange juice as a morning snack. Despite no changes in anthropometric values, lipid panels, insulin, leptin, and inflammatory markers, orange juice contributed to a significant 25% increase in antioxidant status, as determined by total antioxidant status, as well as a marked 50% reduction in lipid peroxidation, as determined by pretest and post-test malondialdehyde concentrations. The nutrient content of orange juice most likely contributed to the effect observed in improving antioxidant status and suppressing lipid peroxidation and is consistent with our earlier human and animal studies.23,38 This study disagrees with those who reported that drinking 20 fl oz of blood orange juice has no effect on the total antioxidant capacity and malondialdehyde concentrations.47 The discrepancy may be related to the duration of the study. In our study,
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subjects drank freshly squeezed orange juice for 3 months, whereas in the study conducted by Riso et al., subjects drank blood orange juice for 3 weeks.47 However, this study agrees with those who reported that drinking *6.7 fl oz of orange juice with one apple and a pear daily for 26 days increased the plasma total antioxidant capacity.48 Moreover, decreased lipid peroxidation evident from drinking orange juice is associated with increased antioxidant status independent of inflammatory markers, hormones, or cholesterol and triglyceride levels. Similar to our previous carrot juice study, drinking orange juice did not affect plasma insulin, leptin, IGF-1, interleukin1a, homocysteine, and C-reactive protein, which may have been because laboratory values were all within normal ranges like the beginning of the study.23 We hypothesize that high mean cholesterol and triglyceride concentrations observed in this study are actually attributed to the eating habits and lifestyle factors. In study conducted by Alvarez-Parrilla,48 subjects consumed one apple, one pear, and *6.7 fl oz of orange juice for 26 days, and their total cholesterol, HDL cholesterol, and LDL cholesterol increased significantly, but the increase in concentrations did not exceed the normal range set by the health authorities.48 Discrepancy in results reported in this study compared with an earlier report may be related to the duration of the study, age of the subjects, combination of eating three fruits containing fiber, and overall eating habits. As expected, there were physiological differences that existed between male and female participants. As anticipated and reported in other studies, men weighed more and had a lower body fat percentage than their female counterparts.23,49,50 In contrast, female participants had higher HDL and higher leptin concentrations. The higher plasma leptin and HDL levels in females agree with an earlier report and are attributed to sex hormones, as observed in another study.23 However, elevated plasma homocysteine and malondialdehyde observed in men suggest that they are at a greater risk for developing CVD than women participants and it may partly be related to men experiencing higher levels of oxidative stress than women. In conclusion, drinking 20 fl oz of freshly squeezed orange juice daily significantly increased antioxidant status and reduced malondialdehyde, which is a marker for lipid peroxidation, without affecting plasma cholesterol and triglyceride status.
ACKNOWLEDGMENTS This project is based upon work supported by the USDACSREES nos. 2008-34402-19195 and 2010-34402-20875 ‘‘Designing Foods for Health’’ through the Vegetable & Fruit Improvement Center. AUTHOR DISCLOSURE STATEMENT The authors declare that they have no competing interests. REFERENCES 1. American Heart Association: Heart Disease and Stroke Statistics. 2005; http://my.clevelandclinic.org/Documents/heart/1105390918119 HDSStats2005Update.pdf (accessed December 2012).
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