assessment of human carotenoid status using raman spectroscopy

Assessment of Human Carotenoid Status Using

Raman Spectroscopy

Presented by:

Angela Mastaloudis, Ph.D.Pharmanex Research Institute

Provo, UT, USA

Carotenoids

An important group of phytonutrients Abundant in fruits and vegetables Epidemiological and clinical studies indicate

protection against the development of a variety of chronic diseases Lutein and zeaxanthin

eye health -carotene and lycopene

protection from sun damage Lutein and lycopene

cardiovascular health Lycopene

prostate cancer

Carotenoids Safe sources of vitamin A

- & -carotene

Fat-soluble antioxidants 1O2 quenchers

Promote immune function -carotene

Protect cellular DNA lycopene

Protect macular region of retina lutein

Lycopene

-carotene

-carotene

Zeaxanthin

Lutein

Relevance of Accurate Assessment of Human Carotenoid Status

• Marker of oxidative stresssmoking, sunlight exposure, pollution all influence

skin carotenoids independent of dietary intake

• Survey fruit and vegetable consumption• Monitor compliance to diets

rich in fruits and vegetables• Monitor intake of carotenoid

containing dietary supplements

Resonant Raman Scattering of Carotenoids

Is = N(Ei) R(if) IL

Scattered Intensity

Configuration Coordinate

1 Ag1E

nerg

y

1 Bu1

Configuration Coordinate

1 Ag1E

nerg

yA

bsor

ptio

n

Abs

orpt

ion

2 Ag1

1 Bu1

Configuration Coordinate

1 Ag1E

nerg

yA

bsor

ptio

n

Abs

orpt

ion

2 Ag1

1 Bu1

Configuration Coordinate

1 Ag1

forb

idde

nlu

m.

Ene

rgy

wea

klu

m.

Abs

orpt

ion

Abs

orpt

ion

Lum

inescence

2 Ag1

1 Bu1

Configuration Coordinate

1 Ag1

forb

idde

nlu

m.

Ene

rgy

wea

klu

m.

Abs

orpt

ion

Abs

orpt

ion

Ram

ansc

atte

ring

Lum

inescence

Courtesy of W. Gellermann

Ei Ef

hLhR

Resonant Raman Scatteringof Carotenoids

8 0 0 1 2 0 0 1 6 0 0Ram an Shif t / cm

- 1

1 5 2 85

cm- 1

1 1 5 89

cm- 1

C- C

O H

O H

C- C

C= C

C-CH3

f ilt er paper

Courtesy of W. Gellermann

Early Studies: The Eye & AMD

Macula

L & Z

Gellermann W, Ermakov IV, McClane RW, Bernstein PS. Raman imaging of human macular pigments. Optics Letters. 2002;27:833

Courtesy of W. Gellermann

BioPhotonic Scanner

• 473 nm excitation, 511 nm detection (C=C)

Carotenoids are measured at the palm of the hand in the top 0.1 mm of the stratum corneum layer.

Skin Carotenoids

• Carotenoids are measured in the stratum corneum layer of the skin

• Carotenoids protect epidermis from UV Light

• Site of action advantage

Skin Carotenoids

• An Important Indicator of the Body’s Antioxidant Defense System

• Most convenient measure of antioxidant status• Carotenoids measured:

-Carotene, -Carotene, Lycopene, Lutein, Zeaxanthin, -Cryptoxanthin

First line of defense & part of antioxidant network• Skin carotenoids are influenced by oxidative stress

(smoking, sunlight exposure, pollution) independent of dietary intake

Advantages

• Non-invasive

• Safe

• Instant results

• Inexpensive Highly specific

• Reflects long-term carotenoid status

Clinical validation of a non-invasive, Raman spectroscopic method to assess carotenoid nutritional status in humans

Jeffrey A. Zidichouski1,3, Angela Mastaloudis1, Stephen J. Poole1, James C.

Reading2 and Carsten R. Smidt1

1Pharmanex Research Institute, Provo, Utah, USA; 2U. of Utah School of Medicine, SLC, Utah, USA; 3Institute for

Nutrisciences and Health, NRC, Canada

Background

• Serum/plasma carotenoids (HPLC)currently accepted “gold standard”used to validate subjective fruit & vegetable intake

data (epidemiological research)used to assess human carotenoid and antioxidant

status invasive & labor-intensivenot suitable for large populations

Objectives

• To validate the Raman spectroscopy (RS) methodology (BioPhotonic Scanner) by comparing it to HPLC, the currently accepted gold standard for assessing human carotenoid status using Criterion Validity

• To assess and compare reliability of the two measures

Methodology

• 372 adult non-smokers

• 3 measurements within 8 days, ≥ 48 h apart, after overnight (12 h) fast:Serum carotenoids by HPLC Skin carotenoids, Raman spectroscopyDemographic data and food frequency questionnaires

Results

Carotenoids (means ± SD) Serum

(g/ml) Raman Intensity

(Counts)

N = 372 1.10 ± 0.51 20,102 ± 6,386

•Reliability (Intra-Subject Variability)Skin (Raman Spec.): 8.2 %*Serum (HPLC): 8.9 %

*significant difference between skin and serum carotenoids (p < 0.05)

Skin-Serum Correlation

R = 0.81; p < 0.001

Conclusions

• Raman spectroscopy (BioPhotonic Scanner) is a valid measure to assess skin carotenoid status in situ in humans

• RS accurately predicts blood serum total carotenoids, independent of demographic and lifestyle factors

• Skin carotenoids are more stable over time than serum carotenoids, likely because they are not confounded by recent dietary intake (Dueker SR et al. J Lipid Res. 2000;41:1790)

• RS appears to be a better indicator of carotenoid status than blood carotenoids

Epidemiological Studies

2004 Data — 33,000 SubjectsF

req

ue

ncy Antioxidant Supplementation

5-9 Fruits & Vegetables

Average (2-3 Fruits & Vegetables)Smokers

Obesity

Antioxidant Supplementation+ 5-9 Fruits & Vegetables

Ramen Intensity, Counts

Fruit & Vegetable Intake

31,100

28,560

24,773

21,981

10,000

15,000

20,000

25,000

30,000

35,000

1 or Less 2 to 3 4 to 5 6 or More

Reported Daily Fruit & Vegetable Servings

Ram

an

In

ten

sit

y, C

ou

nts

.

n = 9,336 n = 13,600 n = 6,916 n = 2,796

p < 0.01 p < 0.01 p < 0.01

2004 Data of 32,648 Non-Supplement Users

Supplementation: LP**Multivitamin/mineral Supplement with Antioxidant Nutrients

25,39026,236

34,718

29,672

10,000

15,000

20,000

25,000

30,000

35,000

40,000

None Irregular One/Day Two/Day

Reported LP Usage

Ram

an I

nte

nsi

ty,

Co

un

ts

.

n = 4,014n = 1,361n = 958n = 32,941

p < 0.01 p < 0.01 p < 0.01

2004 Data of 39,656 Subjects

Body Mass Index

29,236

25,612

20,835

16,603

10,000

15,000

20,000

25,000

30,000

35,000

< 25 25-29 30-39 > 40

Body Mass Index (BMI, kg/m2)

Ram

an

In

tesit

y, C

ou

nts

.

n = 11,588 n = 8,475 n = 422

p < 0.01 p < 0.01 p < 0.01

n = 4,550

2004 Data of 25,035 Subjects

Smoking (Oxidative Stress)

19890

17591

13056

11593

0

5000

10000

15000

20000

25000

Non-Smokers < 1/day 1-5/day > 5/day

n = 1047 n = 10 n = 16 n = 32

Ram

en I

nte

nsi

ty,

Co

un

ts

Urinary MDA Test

Scanner Readings vs. Urinary MDA(means +/- SD)

1939222025 23047

29590

0

5000

10000

15000

20000

25000

30000

35000

40000

High Medium Low Optimum

Urinary MDA Test Results

n = 4

n = 51n = 490

n = 17

Correlation: r = - 0.135, p < 0.01

Ram

en In

ten

sity

, Co

un

ts

Epidemiological Studies Summary

Skin carotenoids:Are not influenced by age, gender or race Increase linearly in response to both dietary

intake and intake from supplementsAre inversely correlated with oxidative stress

markers(smoking, UV light exposure, urinary F2-Isoprostanes)

Are a valid indicator of overall antioxidant status

Randomized, Placebo Controlled Antioxidant Supplementation Study

Study Design

• N = 52 • RCT: Twice a day, with breakfast and with dinner,

subjects took LP nano or Placebo• Study duration: 18 weeks• BioPhotonic Scanner

Subjects were scanned a total of 15 times

• Lifestyle Subjects encouraged to continue with their typical diet and

exercise habits for the entire study Diet & Lifestyle Questionnaires

Scanner Scores Increased Rapidly

*p<0.05 compared to wk 0 #p<0.05 compared to placebo

#, *#, *

#, *

#, *#, *

#, *#, *#, *#, *

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

0 1 2 3 4 6 8 10 13 15 18

Weeks

Sca

nn

er

Sco

re (

Co

un

ts)

LifePak Nano Placebo

N = 42

LP Nano

Summary

• Skin carotenoids increased significantly with supplementation ( two weeks)

• Skin carotenoids increased in all 20 subjects in twenty weeks with supplementation

• Even after 18 weeks, skin carotenoids were still increasing with supplementation

Double-Blind, Placebo-Controlled Antioxidant Supplementation Study

• RLI, Florida Hospital Celebration Health

• 53 subjects who had not taken antioxidant supplements within the last 3 months

• 6 weeks on supplements (LP or Placebo)

• Measured skin RS response and serum antioxidants at days 0, 21 and 42

Double-Blind, Placebo-Controlled Antioxidant Supplementation Study

Baseline Subject Characteristics

(means ± SD) Supplement Placebo N (m/f) 25 (5/20) 28 (4/24) Age (y) 54.6 ± 5.0 56.1 ± 10.5 Weight (kg) 72.7 ± 14.7 71.2 ± 13.7 BMI (kg/m2) 26.9 ± 4.8 26.6 ± 5.0 Tot. Cholesterol (mg/dl) 231 ± 41 224 ± 59 Blood Glucose (mg/dl) 94.3 ± 9.0 95.4 ± 9.2

Results: Serum Carotenoids

0

500

1000

1500

2000

2500

3000

Day 0 Day 21 Day 42

Ser

um

Car

ote

no

ids

(ng

/ml)

SupplementPlacebo

p<0.05p<0.05

Means ± S.D.

Results: Skin Carotenoids

10000

15000

20000

25000

30000

35000

Day 0 Day 21 Day 42

Ram

an In

ten

sity

SupplementPlacebo

p<0.05

Means ± S.D.

Results: Serum Vitamins C and E

0

5

10

15

20

25

Day 0 Day 21 Day 42

Asc

orb

ic A

cid

(m

cg/m

l)

SupplementPlacebo

0

5

10

15

20

25

30

35

Day 0 Day 21 Day 42

Alp

ha-

To

cop

her

ol (

mcg

/ml) Supplement

Placebo

p<0.05 p<0.05

p<0.05

Can Skin Carotenoids Serve as a Marker of Antioxidant Status or

Oxidative Stress?

THIOLCycle

(GSH or LA)VITAMIN Ccycle

Lipid–water interface

Sunlight, energy metabolism, strenuous exercise, cigarette smoke, pollution

O·2– and other free radicals

VITAMIN Ecycle

ROOHROH

PUFA

ROO•RO•

The Antioxidant Network

NAD(P)H + H+

NAD(P)+

Carotenoids

Associations of Antioxidant Status and Oxidative Stress with Skin Carotenoids Assessed by

Raman SpectroscopyJoseph Carlson1,3, Shayn Stavens1,

Richard Holubkav1, Jeffrey Zidichouski2, Angela Mastaloudis2, Carsten Smidt2, Eldon Askew1

1U. of Utah Salt Lake City, Utah; 2Pharmanex Research Institute Provo, UT; 3Michigan State U. East Lansing, MI

Objectives

• To evaluate the relationship between skin carotenoids and serum antioxidants (vitamins E and C)

• To evaluate the relationship between carotenoid antioxidant status and markers of oxidative stress

Study Design

• N = 307 adult non-smokers

• Serum carotenoids, vitamin E and vitamin C by HPLC

• Skin carotenoids using Raman spectroscopy

• Questionnaires: demographic, lifestyle and dietary data

• Urinary F2-Isoprostanes

F2-Isoprostanes• Gold standard for measurement of in vivo lipid

peroxidation• Reliable • Chemically stable• Specific end-product of the free radical mediated oxidation

of arachidonic acid (20:4)• Possess biological pro-atherogenic activity

Potent vasoconstrictors Cause platelet aggregation

• Plasma concentrations are elevated in subjects with known oxidative stress (smokers, diabetics, obesity, cardiovascular disease, Alzheimer’s Disease etc)

Skin and Serum Carotenoids are Strongly Correlated

Scanner Score

0 10,000 20,000 30,000 40,000 50,000 60,000

0

1.0

2.0

3.0

4.0

Ser

um

Car

ote

no

ids

(M

)

R = 0.81; p < 0.0001

Skin Carotenoids are Positively Correlated with Plasma Antioxidants

Scanner Score

0 10,000 20,000 30,000 40,000 50,000 60,000

10

20

30

40

50

60

70

Pla

sma

An

tio

xid

ants

(M

) R = 0.321; p < 0.001

Skin Carotenoids are Inversely Correlated with F2-Isoprostanes

0 10,000 20,000 30,000 40,000 50,000 60,000

Scanner Score

0

2.0

4.0

6.0

8.0

Uri

nar

y F

2-I

sop

rost

anes

(p

mo

l/L

)

R = 0.23; p < 0.0001

Summary

• Skin carotenoids assessed by the Biophotonic Scanner were directly correlated with:Serum CarotenoidsPlasma antioxidants (Vitamins E and C)Fruit and vegetable intake

• Skin carotenoids were inversely related to F2-Isoprosanes, a marker of oxidative stress

Taiwan Data (n = 38,843; April 2006)

n = 20181 n = 14882 n = 3132 n = 648

Fruit & Vegetable Consumption

Ram

an I

nte

nsi

ty,

Co

un

ts

0

5,000

10,000

15,000

20,000

25,000

30,000

<2 servings 2-3 servings 4-5 servings 6+ servings

Taiwan Data (n = 38,942; April 2006)

n = 111 n = 1546 n = 783 n = 248 n = 12

n = 6383 n = 24529 n = 6881 n = 1149

Body Mass Index (kg/m2)

Ram

an I

nte

nsi

ty,

Co

un

ts

0

5,000

10,000

15,000

20,000

25,000

30,000

14 - 18 19 - 24 25 - 29 30 - 39

Taiwan Data (n = 38,907; April 2006)

Smoking Statusn = 1796 n = 31023 n = 6088

Ram

an I

nte

nsi

ty,

Co

un

ts

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

Former No Yes

Conclusions

Measurement of skin carotenoids using Raman spectroscopy (BioPhotonic Scanner) is: highly correlated with serum carotenoids less variable than serum carotenoidspreferable to serum carotenoids as a marker of

fruit and vegetable intakeReflective of long-term carotenoid status indicative of other serum antioxidants (vitamins C and E) indicative of oxidative stress (MDA,F2-Isoprostanes)

Summary

• Resonance Raman scattering is a viable optical technique to measure skin carotenoids in vivo. It is highly specific, non-invasive and suitable for clinical field measurements of large populations.

Acknowledgments • Stephen Poole, Carsten Smidt – Pharmanex Research Institute

• Jason Morrow – Vanderbilt University

• Neal Craft – Craft Technologies, NC

• Lester Packer – University of Southern California

• Kyung-Jin Yeum, Jeff Blumberg – Tufts University

• James Rippe – Rippe Lifestyle Institute

• Wayne Askew, Joe Carlson, Shayn Stavens University of Utah, Division of Foods and Nutrition, SLC

• Jeff Zidichouski – Canadian National Research Council (P.E.I.)

• Werner Gellermann – University of Utah, Physics Dept., SLC