Assessment of Human Carotenoid Status Using Raman Spectroscopy

1
Assessment of Human Carotenoid Status Using Raman
Spectroscopy

• Presented by
• Angela Mastaloudis, Ph.D.
• Pharmanex Research Institute
• Provo, UT, USA

2
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

3
Carotenoids

• Safe sources of vitamin A
• a- b-carotene
• Fat-soluble antioxidants
• 1O2 quenchers
• Promote immune function
• b-carotene
• Protect cellular DNA
• lycopene
• Protect macular region of retina
• lutein

Zeaxanthin
Lutein
4
Relevance of Accurate Assessment of Human
Carotenoid Status

• Marker of oxidative stress
• smoking, 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

5
Resonant Raman Scattering of Carotenoids
6
Resonant Raman Scatteringof Carotenoids
Courtesy of W. Gellermann
7
Early Studies The Eye AMD
L Z
Macula
Gellermann W, Ermakov IV, McClane RW, Bernstein
PS. Raman imaging of human macular pigments.
Optics Letters. 200227833
Courtesy of W. Gellermann
8

• BioPhotonic Scanner
• 473 nm excitation, 511 nm detection (CC)

Carotenoids are measured at the palm of the hand
in the top 0.1 mm of the stratum corneum layer.
9
Skin Carotenoids

• Carotenoids are measured in the stratum corneum
  layer of the skin
• Carotenoids protect epidermis from UV Light
• Site of action advantage

10
Skin Carotenoids

• An Important Indicator of the Bodys Antioxidant
  Defense System
• Most convenient measure of antioxidant status
• Carotenoids measured
• a-Carotene, b-Carotene, Lycopene, Lutein,
  Zeaxanthin, b-Cryptoxanthin
• First line of defense part of antioxidant
  network
• Skin carotenoids are influenced by oxidative
  stress (smoking, sunlight exposure, pollution)
  independent of dietary intake

11
Advantages

• Non-invasive
• Safe
• Instant results
• Inexpensive Highly specific
• Reflects long-term carotenoid status

12
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

13
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-intensive
• not suitable for large populations

14
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

15
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 spectroscopy
• Demographic data and food frequency questionnaires

16
Results

• Carotenoids (means SD)

• Reliability (Intra-Subject Variability)
• Skin (Raman Spec.) 8.2
• Serum (HPLC) 8.9
• significant difference between skin and serum
  carotenoids (p lt 0.05)

17
Skin-Serum Correlation

• R 0.81 p lt 0.001

18
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. 2000411790)
• RS appears to be a better indicator of carotenoid
  status than blood carotenoids

19
Epidemiological Studies
20
2004 Data 33,000 Subjects
Ramen Intensity, Counts
21
Fruit Vegetable Intake
22
Supplementation LPMultivitamin/mineral
Supplement with Antioxidant Nutrients
40,000
2004 Data of 39,656 Subjects
35,000
34,718
30,000
29,672
Raman Intensity, Counts .
26,236
25,000
25,390
p lt 0.01
p lt 0.01
p lt 0.01
20,000
15,000
n 4,014
n 1,361
n 958
n 32,941
10,000
None
Irregular
One/Day
Two/Day
Reported LP Usage
23
Body Mass Index
24
Smoking (Oxidative Stress)
Ramen Intensity, Counts
25
Urinary MDA Test
Ramen Intensity, Counts
26
Epidemiological Studies Summary

• Skin carotenoids
• Are not influenced by age, gender or race
• Increase linearly in response to both dietary
  intake and intake from supplements
• Are inversely correlated with oxidative stress
  markers
• (smoking, UV light exposure, urinary
  F2-Isoprostanes)
• Are a valid indicator of overall antioxidant
  status

27
Randomized, Placebo Controlled Antioxidant
Supplementation Study
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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

29
Scanner Scores Increased Rapidly
LP Nano

plt0.05 compared to wk 0 plt0.05 compared to
placebo
,
,
N 42
,
,
,
,
,
,
,
30
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

31
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

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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
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Results Serum Carotenoids
Means S.D.
plt0.05
plt0.05
34
Results Skin Carotenoids
Means S.D.
plt0.05
35
Results Serum Vitamins C and E
plt0.05
plt0.05
plt0.05
36
Can Skin Carotenoids Serve as a Marker of
Antioxidant Status or Oxidative Stress?
37
The Antioxidant Network
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Associations of Antioxidant Status and Oxidative
Stress with Skin Carotenoids Assessed by
Raman Spectroscopy

• Joseph 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

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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

40
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

41
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 (204)
• 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, Alzheimers
  Disease etc)

42
Skin and Serum Carotenoids are Strongly
Correlated
R 0.81 p lt 0.0001
Serum Carotenoids (mM)
Scanner Score
43
Skin Carotenoids are Positively Correlated with
Plasma Antioxidants

R 0.321 p lt 0.001
Plasma Antioxidants (mM)
Scanner Score
44
Skin Carotenoids are Inversely Correlated with
F2-Isoprostanes

8.0
R 0.23 p lt 0.0001
6.0
Urinary F2-Isoprostanes (pmol/L)
4.0
2.0
Isoprostanes pmol / L
0
0
10,000
20,000
30,000
40,000
50,000
60,000
Scanner Score
45
Summary

• Skin carotenoids assessed by the Biophotonic
  Scanner were directly correlated with
• Serum Carotenoids
• Plasma antioxidants (Vitamins E and C)
• Fruit and vegetable intake
• Skin carotenoids were inversely related to
  F2-Isoprosanes, a marker of oxidative stress

46
Taiwan Data (n 38,843 April 2006)
Fruit Vegetable Consumption
n 14882
n 3132
n 20181
n 648
30,000
25,000
20,000
Raman Intensity, Counts
15,000
10,000
5,000
0
lt2 servings
2-3 servings
4-5 servings
6 servings
47
Taiwan Data (n 38,942 April 2006)
Body Mass Index (kg/m2)
30,000
n 6383
n 24529
n 6881
n 1149
25,000
20,000
Raman Intensity, Counts
15,000
10,000
5,000
n 111
n 1546
n 783
n 248
n 12
0
14 - 18
19 - 24
25 - 29
30 - 39
48
Taiwan Data (n 38,907 April 2006)
Smoking Status
35,000
n 1796
n 31023
n 6088
30,000
25,000
20,000
Raman Intensity, Counts
15,000
10,000
5,000
0
Former
No
Yes
49
Conclusions

• Measurement of skin carotenoids using Raman
  spectroscopy (BioPhotonic Scanner) is
• highly correlated with serum carotenoids
• less variable than serum carotenoids
• preferable to serum carotenoids as a marker of
  fruit and vegetable intake
• Reflective of long-term carotenoid status
• indicative of other serum antioxidants (vitamins
  C and E)
• indicative of oxidative stress (MDA,F2-Isoprostane
  s)

50
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.

51
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