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 L y c o p e n a - c r o t e n b - c a r o t e n
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
Ei Ef
hL
hR
Courtesy of W. Gellermann
Is = N(Ei) sR(if) IL
Scattered Intensity

6. Resonant Raman Scattering of Carotenoids
Carotenoids have a characteristic Raman spectral fingerprint generated from vibrations of their long carbon backbone
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. 2002;27:833
Courtesy of W. Gellermann

8. 473 nm excitation, 511 nm detection (C=C)
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.

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 Body’s 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)
Serum ( m
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)

17. Skin-Serum Correlation
R = 0.81; p < 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. 2000;41:1790)
RS appears to be a better indicator of carotenoid status than blood carotenoids

19. Epidemiological Studies

20. Ramen Intensity, Counts
2004 Data — 33,000 Subjects
Frequency
Antioxidant Supplementation
5-9 Fruits & Vegetables
Average (2-3 Fruits & Vegetables)
Smokers
Obesity
+ 5-9 Fruits & Vegetables
Ramen Intensity, Counts

21. Fruit & Vegetable Intake

22. Raman Intensity, Counts .
Supplementation: LP* *Multivitamin/mineral Supplement with Antioxidant Nutrients
40,000
2004 Data of 39,656 Subjects
35,000
34,718
30,000
29,672
Raman Intensity, Counts .
25,000
26,236
25,390
p < 0.01
p < 0.01
p < 0.01
20,000
15,000
n = 32,941
n = 958
n = 1,361
n = 4,014
10,000
None
Irregular
One/Day
Two/Day
Reported LP Usage

23. Body Mass Index
There is an interesting link between body fat levels and scanner scores - as body fat levels increase, scanner scores decrease.

24. Smoking (Oxidative Stress)
Ramen Intensity, Counts

25. Ramen Intensity, Counts
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

28. 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
*p<0.05 compared to wk 0 #p<0.05 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

32. 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

33. Results: Serum Carotenoids
Means ± S.D.
p<0.05
p<0.05

34. Results: Skin Carotenoids
Means ± S.D.
p<0.05

35. Results: Serum Vitamins C and E
p<0.05
Results: Serum Vitamins C and E
p<0.05
p<0.05

36. Can Skin Carotenoids Serve as a Marker of Antioxidant Status or Oxidative Stress?

37. The Antioxidant Network
VITAMIN C cycle
Lipid–water
interface
NAD(P)H + H+
NAD(P)+
THIOL
Cycle
(GSH or a-LA)
VITAMIN E
cycle
ROOH
ROH
PUFA
ROO•
RO•
Carotenoids
Sunlight, energy metabolism, strenuous exercise, cigarette smoke, pollution
O·2– and other free radicals

38. 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

39. 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 (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)

42. Skin and Serum Carotenoids are Strongly Correlated
10,000
20,000
30,000
40,000
50,000
60,000
1.0
2.0
3.0
4.0
Serium Carotenoids uM
R = 0.81; p <
Serum Carotenoids (mM)
Scanner Score

43. Skin Carotenoids are Positively Correlated with Plasma Antioxidants10 20 30 40 50 60 70
Total Antioxidants
R = 0.321; p < 0.001
Plasma Antioxidants (mM)
10,000
20,000
30,000
40,000
50,000
60,000
Scanner Score

44. Skin Carotenoids are Inversely Correlated with F2-Isoprostanes
8.0
R = 0.23; p <
6.0
Urinary F2-Isoprostanes (pmol/L)
4.0
2.0
Isoprostanes pmol / L
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 = 20181
n = 14882
n = 3132
n = 648
30,000
25,000
20,000
Raman Intensity, Counts
15,000
10,000
5,000
<2 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

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
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-Isoprostanes)

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