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 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

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

Resonant Raman Scattering of Carotenoids Ei Ef hL hR Courtesy of W. Gellermann Is = N(Ei) sR(if) IL Scattered Intensity

Resonant Raman Scattering of Carotenoids Carotenoids have a characteristic Raman spectral fingerprint generated from vibrations of their long carbon backbone Courtesy of W. Gellermann

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

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.

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: 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

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-intensive not 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 spectroscopy Demographic data and food frequency questionnaires

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)

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

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

Fruit & Vegetable Intake

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

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

Smoking (Oxidative Stress) Ramen Intensity, Counts

Ramen Intensity, Counts Urinary MDA Test Ramen Intensity, Counts

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

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 LP Nano *p<0.05 compared to wk 0 #p<0.05 compared to placebo #, * #, * N = 42 #, * #, * #, * #, * #, * #, * #, *

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 Means ± S.D. p<0.05 p<0.05

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

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

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

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

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

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 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 < 0.0001 Serum Carotenoids (mM) Scanner Score

Skin Carotenoids are Positively Correlated with Plasma Antioxidants 10 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

Skin Carotenoids are Inversely Correlated with F2-Isoprostanes 8.0 R = 0.23; p < 0.0001 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

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

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

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

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

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)

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