Daily Consumption of Grapefruit for 6 Weeks Reduces Urine F2-Isoprostanes in Overweight Adults with High Baseline Values but Has No Effect on Plasma High-Sensitivity

Daily Consumption of Grapefruit for 6 Weeks

Reduces Urine F2-Isoprostanes in Overweight Adults with High Baseline

Values but Has No Effect on Plasma High-Sensitivity C-Reactive Protein or Soluble Vascular Cellular Adhesion

Molecule 1

Denise A. PalmaNFS5331 Seminar

Spring 2014 Dr. Radcliffe

(Ref. 1, p.1)

Atherosclerosis Metabolic Syndrome Antioxidants Background and Purpose of the Study Hypothesis Description of participants Statistical Methods and Parameters Results and Conclusion Recommendations for future research References

Overview

Atherosclerosis (AS) Injury to the endothelial

cells that line artery walls resulting in formation of plaque (2)

Cardiovascular disease (CVD) is the leading cause of death in the United States (3).

Over 12 million individuals are affected by AS resulting in more than one half million deaths each year (3).

Common risk factors (2) Smoking Hypertension Diabetes Increased LDL Decreased HDL

http://www.nhlbi.nih.gov/health/health-topics/images/ather_lowres.gif

(Ref. 2, p. 618; Ref.3, p.298-299)

Underlying risk factors Obesity, physical inactivity, atherogenic diet

(4). Major risk factors

Cigarette smoking, hypertension, elevated LDL-C and low HDL-C, family history of coronary heart disease (CHD) and aging (4).

Emerging risk factors Elevated trigylcerides TGs, small LDL

particles, insulin resistance, glucose intolerance, proinflammatory state and prothrombotic state (4).

Metabolic Syndrome (MetS)

(Ref.4, p.432)

Clinical Measure NCEP

Insulin Resistance • None but any three of the following five

Body Weight • WC ≥ 102 cm in men OR ≥ 88 cm in women

Lipid • TG ≥ 150 mg/dL• HDL-C < 40 mg/dL in men OR <

50 mg/dL in women

Blood pressure • ≥ 130/85 mm/Hg

Glucose • ≥ 100 mg/dL (includes diabetes)

Metabolic Syndrome (MetS)

NCEP, National Cholesterol Education Program Adult Treatment Panel III;

WC, waist circumference; BMI, body mass index; TG, triglycerides; HDL-C, HDL cholesterol.(Ref.1, p. 1587, Ref.4, p.433,Ref.5, p.630)

Phytochemicals act as antioxidants 1. Scavenge free radicals causing

oxidative stress (6). 2. Suppress reactive oxygen species

(ROS) (7). Flavanoids

Flavanones- found in citrus fruits (7) hesperetin and naringenin

Prevent human disease or delay oxidative stress (8).

Antioxidants

(Ref. 6, p.703-707, Ref. 7, p. 1036-1037, Ref. 8, p.260-262

Background and Purpose of the Study

Previous studies suggest both citrus and grapefruit may promote cardiovascular health

• Prospective Epidemiological Study of Myocardial Infarction (PRIME)• Reduction in ischemic events

• Nurse’s Health Study (NHS)• Reduced concentrations of fasting

proinflammatory CVD cytokines

Same research group previously demonstrated positive results

in healthy, overweight and obese adults after consuming Rio red grapefruit daily for six (6) weeks. (Ref.1, p.1587)

Consumption of daily Rio red grapefruit intake before each meal assists in reduction of atherosclerotic plaque production by reducing (1): Soluble vascular adhesion molecule 1

(sVCAM-1) High-sensitivity C-reactive protein

(hsCRP) F2-Isoprostanes

Hypothesis

(Ref.1, p. 1587)

85 nonsmoking men and premenopausal women

>18 yrs Free of inflammatory disease BMI between 25 and 45 kg/m²

Description of participants

(Ref.1, p. 1587)

Grapefruit Groupn = 37

Control Groupn = 32

Average Age (years)

40.6 ± 10.8 43.2 ± 10.6

Female n= 25 n = 23

Male n = 12 n = 9

Weight kg (lb) 92.1 ± 15.0 (202 lb) 90.8 ± 12.8 (199 lb)

AVerage BMI (kg/m2)

32.8 ± 4.2 31.4 ± 3.8

Metabolic Syndrome

n = 14 n = 15

Description of participants

(Ref.1, p. 1587-1588)

Baseline washout for 3 weeks restricting fruit and vegetables high in polyphenol and carotenoid (1).

Urine and blood samples collected at the end of washout period and intervention period.

Height, weight, and waist circumference (WC) measured at baseline, wk 6 and wk 9

69 participants divided into two groups: Grapefruit group (n = 37) continued on low

bioactive diet with a 3x daily ate a half a grapefruit for 6 wks.

Control group (n = 32) continued on low bioactive diet for 6 wks.

Methods: Collection

(Ref.1, p. 1587)

Randomized controlled trial Chi-square (x²) used to compare categorical variables. t test to compare continuous variables.

Urine and blood samples collected at end of washout period (3 wks) and end of intervention period (9 wks) Paired t test used to compare the before and after effects. Linear Regression used to further predict the relationship

between an independent variable and a dependent variable. Stratified Analysis

Analysis of participants with MetS utilized the same method mentioned above (n = 29).

Height, weight, and Waist Circumference (WC) measured at baseline, wk 6 and wk 9 Linear regression used to assess the CVD risk factors and

biomarkers.

Method: Analysis

(Ref.9 and Ref.10)

Chi-square (x²) A chi-square test is used to compare when only two categorical variables exist .

t test A t test is used to compare two continuous variables by utilizing their means.

Paired t testA paired t test is utilized to compare the

before and after effects by evaluating the means of the continuous variables.

Linear RegressionA linear regression is used to further predict

the relationship between an independent variable and a dependent variable.

Stratified Analysis Stratified analysis further categorizes results in order to view further correlation followed by a paired t test within each new category

to view any statistical significance.

Statistical Methods and Parameters

(Ref.9 and Ref.10)

BeforeIntervention

AfterIntervention

P-value¹

Grapefruit 628 ± 117

614 ± 123

P = 0.11

Control 623 ± 125

611 ± 111

P = 0.26

Soluble vascular adhesion molecule 1 (sVCAM-1)²

Mean Plasma sVCAM-1 ug/L

¹ Paired t test used to compare the before and after effects.² Soluble vascular adhesion molecule-1 (1, 11)

(Ref. 1, p.1589, Ref. 11, p. 25-27

Results: Before and After Results for Grapefruit Group

Before After605

610

615

620

625

630

Mean Plasma sVCAM-1

Pla

sm

a s

VC

AM

-1 u

g/L

P = 0.11

Results: Before and After Results for Control Group

Before After605

610

615

620

625

Mean Plasma sVCAM-1

Pla

sm

a s

VC

AM

-1

ug

/L

P = 0.26

High-sensitivity C-reactive protein (hsCRP)

Low risk of developing

CVD

Lower than 1.0 mg/L

Average risk of developing

CVD

Between 1.0 and 3.0 mg/L

High risk of developing

CVD

Higher than 3.0 mg/L

(Ref. 12. Ref.13, p.C20-21)

High-sensitivity C-reactive protein (hsCRP)

BeforeIntervention

AfterIntervention

P-value¹

Grapefruit 2.2 ± 1.5 2.1 ± 1.5 P = 0.60

Control 2.4 ± 2.0 2.8 ± 2.0 P = 0.14

Mean Plasma hsCRP mg/L

¹ Paired t test used to compare the before and after effect.


Before After2

2.05

2.1

2.15

2.2

2.25

Mean Plasma hsCRP

Pla

sm

a h

sC

RP

mg

/L

P = 0.60


Before After2.22.32.42.52.62.72.82.9

Mean Plasma hsCRP

Pla

sm

a h

sC

RP

mg

/L

P = 0.14

F2-Isoprostanes

BeforeIntervention

AfterIntervention

P-value

Grapefruit 14.1 ± 7.7 12.4 ± 6.4 P = 0.09

Control 14.7 ± 6.5 15.9 ± 9.0 P = 0.45

Mean Urinary F2-Isoprostanes ng/mg

¹ Paired t test used to compare the before and after effects.² F2-Isoprostanes (14, 15)

(Ref.14, p.14P-15P, Ref.15, p.1791-1794)


Before After11.5

1212.5

1313.5

1414.5

Mean Urinary F2-Isoprostanes

Uri

nary

F2

-Iso-

pro

sta

nes n

g/m

g

P = 0.09


Before After14

14.5

15

15.5

16


Uri

nary

F2

-Iso-

pro

sta

nes n

g/m

g

P = 0.45

Linear Regression Utilized to Compare Post intervention¹

Biomarker Post-Grapefruit Post-Control P value¹

Plasma sVCAM-1

614 ug/L 611 ug/L P = 0.35

Plasma hsCRP

2.1 mg/L 2.8 mg/L P = 0.09

Urinary F2-

isoprostanes

12.4 ng/mg 15.9 ng/mg P = 0.16

¹ Linear Regression used to further predict the relationship between an independent variable

and a dependent variable.

Stratified analysis further categorizes results in order to view further correlation followed by a paired t test within each new category to view any statistical significance.

Results were further stratified into LOW and HIGH categories for each biomarker to view any additional significance.

Results: Stratification

(Ref.1, p. 1588)


Before/ After LOW <611 Before/After HIGH ≥611560580600620640660680700720

Mean Plasma sVCAM-1

Pla

sm

a s

VC

AM

-1 u

g/L

P = 0.70


P = 0.10

Before/ After LOW <2.18 Before/After HIGH ≥2.180

1

2

3

4

Mean Plasma hsCRP

Pla

sm

a h

sC

RP

mg

/L


P = 0.004

Before/ After LOW <12.7

Before/After HIGH ≥12.705

10152025


Uri

ne F

2-I

so-

pro

sta

nes n

g/m

g

Results: Subsample of Participants with MetS

Biomarker Post-Grapefruit Post-Control P value¹

Plasma sVCAM-1

650 ug/L 594 ug/L P = 0.84

Plasma hsCRP

2.0 mg/L 3.1 mg/L P = 0.11

Urinary F2-

isoprostanes

12.0 ng/mg 18.3 ng/mg P = 0.06

¹ Linear Regression used to further predict the relationship between an independent variable and a dependent variable.

Results: Relationship between Biomarkers and CVD risk factors

Biomarker Baseline Post intervention

Plasma sVCAM-1

Age

Plasma hsCRP

BMI and WC

Urinary F2-isoprostanes

WC

Inversely related in grapefruit arm and

positively associated in control arm

¹ Linear Regression used to further predict the relationship between an independent variable and a dependent variable.

(Ref.1, p. 1588)

Six (6) week intervention of daily grapefruit consumption 3x daily did not reduce sVCAM, hsCRP or F2-isoprostane concentrations.

Slight significance in stratified HIGH baseline and MetS subgroup demonstrated lower F2-isoprostane concentrations post intervention.

Conclusion

(Ref.1, p. 1589)

Participants: Small sample More women than men Provide education to participants on high/low

bioactive diet Self-reported on consuming entire grapefruit

Methods: Limited to only a 6 week period; increase duration Nutritional analysis of fruit

Results: Demonstrated slight improvement in

inflammatory/oxidative response may be an indicator of delay

Review

Increase study size. Utilize different brand of grapefruit with

a nutritional analysis. Attempt with high bioactive diet vs. low

bioactive diet. Utilize participants with healthy BMI to

measure oxidative stress/inflammation biomarkers to view any differences in uptake and outcome.

Recommendations for future research

Dow CA, Wertheim BC, Patil BS, and Thomson CA. Daily Consumption of Grapefruit for 6 Weeks Reduces Urine F2-Isoprostanes in Overweight Adults with High Baseline Values but Has No Effect on Plasma High-Sensitivity C-Reactive Protein or Soluble Vascular Cellular Adhesion Molecule 1. The Journal of nutrition 2013; 1586-1592. NCT01452841

Brashers VL. Alterations of Cardiovascular Funtion. 4th ed. In: Understanding Pathophysiology. St. Louis, MO: Mosby Elsevier, 2008: 607-673.

Pujol TJ, Tucker JE and Barnes JT. Diseases of Cardiovascular System. 2nd. Ed. In: Nutrition Therapy and Pathophysiology. Belmont, CA: Wadsworth Cengage Learning, 2011: 283-339.

References

Grundy SM, Brewer HB., Cleeman JI, Smith SC and Lenfant C. Definition of metabolic syndrome report of the National Heart, Lung, and Blood Institute/American Heart Association Conference on scientific issues related to definition. Circulation 2004; 109(3), 433-438. PMID: 14744958

Grundy SM. Metabolic syndrome pandemic. Arteriosclerosis, thrombosis, and vascular biology 2008; 28(4), 629-636. PMID: 18174459

Kaur C and Kapoor HC. Antioxidants in fruits and vegetables–the millennium’s health. International Journal of Food Science & Technology 2001; 36(7), 703-725. DOI: 10.1111/j.1365-2621.2001.00513.x

References

Pietta PG. Flavonoids as antioxidants. Journal of natural products 2000; 63(7), 1035-1042. PMID: 10924197

Halliwell B. Free radicals and antioxidants: updating a personal view. Nutrition reviews 2012; 70(5):257-65. PMID: 22537212

Green SB, Salkind NJ. Using SPSS for Macintosh and Windows: Analyzing and understanding data. Upper Saddle River, NJ: Prentice Hall, 2011.

References

Caprette D. Experimental Biosciences: Resources for Introductory and Intermediate level laboratory courses. 5 May 2010. Internet: http://www.ruf.rice.edu/~bioslabs/tools/stats/stats.html

Packard RR and Libby P. Inflammation in atherosclerosis: from vascular biology to biomarker discovery and risk prediction. Clinical Chemistry 2008; 54(1), 24-38. PMID: 18160725

Ridker PM, Libby P. Risk Factors for Atherothrombotic Disease. In: Libby P, Bonow RO, Mann DL, Zipes DP, eds. Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine. 8th ed. Philadelphia, Pa; Saunders Elsevier; 2007: chap 39.

References

http://www.ruf.rice.edu/~bioslabs/tools/stats/stats.html

http://www.ruf.rice.edu/~bioslabs/tools/stats/stats.html

Tsimikas S, Willerson JT and Ridker PM. C-reactive protein and other emerging blood biomarkers to optimize risk stratification of vulnerable patients. Journal of the American College of Cardiology 2006; 47(8s1), C19-C31. PMID: 16631506

Montuschi P, Barnes PJ and Roberts LJ. Isoprostanes: markers and mediators of oxidative stress. The FASEB Journal 2004; 18(15), 1791-1800. PMID: 15576482

Tsimikas S. Oxidative biomarkers in the diagnosis and prognosis of cardiovascular disease. The American journal of cardiology 2006; 98(11), S9-S17. PMID: 17126679

References

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Daily Consumption of Grapefruit for 6 Weeks Reduces Urine F2-Isoprostanes in Overweight Adults with High Baseline Values but Has No Effect on Plasma High-Sensitivity