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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 - PowerPoint PPT Presentation
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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