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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

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Assessment of Human Carotenoid Status Using Raman Spectroscopy

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  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. L y c o p e n e a - c a r o t e n e b - c a r o t e n e 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. Ei Ef Scattered Intensity hR Is = N(Ei) sR(if) IL hL Courtesy of W. Gellermann 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. 2002;27:833 Courtesy of W. Gellermann

  8. 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. Serum Raman Intensity (Counts) m ( g/ml) N = 372 1.10 ± 0.51 20,102 ± 6,386 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 < 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. Smokers Average (2-3 Fruits & Vegetables) Obesity 5-9 Fruits & Vegetables Antioxidant Supplementation Frequency Antioxidant Supplementation + 5-9 Fruits & Vegetables 2004 Data — 33,000 Subjects Ramen Intensity, Counts

  21. Fruit & Vegetable Intake

  22. 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

  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

  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

  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 p<0.05 p<0.05

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

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

  38. Associations of Antioxidant Status and Oxidative Stress with Skin CarotenoidsAssessed 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. 4.0 3.0 2.0 1.0 Serium Carotenoids uM 0 0 10,000 20,000 30,000 40,000 50,000 60,000 Skin and Serum Carotenoids are Strongly Correlated R = 0.81; p < 0.0001 Serum Carotenoids (mM) Scanner Score

  43. 70 60 50 40 30 Total Antioxidants 20 10 0 10,000 20,000 30,000 40,000 50,000 60,000 Skin Carotenoids are Positively Correlated with Plasma Antioxidants R = 0.321; p < 0.001 Plasma Antioxidants (mM) Scanner Score

  44. 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 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 <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 15,000 Raman Intensity, Counts 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-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.

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