Carotenoids, Health Benefits and Carotenoids, Health Benefits and

BioavailabilityBioavailabilityCarotenoids, Health Benefits and Carotenoids, Health Benefits and

BioavailabilityBioavailability

Steven J. Schwartz, Ph.D.Steven J. Schwartz, Ph.D.Food Science & Interdisciplinary Graduate Program in NutritionFood Science & Interdisciplinary Graduate Program in Nutrition

The Ohio State UniversityThe Ohio State University

Presented atPresented atPhytochemicals in Fruit and Vegetable to Improve Human HealthPhytochemicals in Fruit and Vegetable to Improve Human Health

November27, 2007November27, 2007

Phytoene

Phytofluene

-Carotene

Neurosporene

Lycopene

Lycopene BiosynthesisLycopene Biosynthesis

Lycopene

-Carotene-Carotene

-Carotene

-Carotene -CaroteneAdapted from Britton, 1983 and Gross, 1991.

Biosynthesis of common Biosynthesis of common ββ and and εε cyclic carotenes cyclic carotenes

Tomatoes Varieties with Unique Carotenoid ProfileTomatoes Varieties with Unique Carotenoid Profile

RED OR1 OR2 YEL GRE

Common CarotenoidsCommon Carotenoids

HydrocarbonsHydrocarbonsXanthophyllsXanthophylls

OH

OH

HO

OH

LuteinLutein

ZeaxanthinZeaxanthin

HO

-Cryptoxanthin-Cryptoxanthin

--CaroteneCarotene

--CaroteneCarotene

LycopeneLycopene

Biological Functions of CarotenoidsBiological Functions of Carotenoids

Provitamin A ActivityProvitamin A Activity

Non-provitamin A Activity:Non-provitamin A Activity:

•• Singlet Oxygen Quenching ActivitySinglet Oxygen Quenching Activity

•• Antioxidant Activity (Trap Free Radicals)Antioxidant Activity (Trap Free Radicals)

• • Enhancement of Immune ResponseEnhancement of Immune Response

•• Potential Chemopreventive PropertiesPotential Chemopreventive Properties

Conversion to Vitamin AConversion to Vitamin A

-carotene-carotene

O2 15,15’-monooxygenase

H+ reductase

retinol

OH

retinol

OH

Carotenoids and Health BenefitsCarotenoids and Health Benefits

EpidemiologicalEpidemiological

Cell cultureCell culture

Animal (experimental)Animal (experimental)

Human (clinical)Human (clinical)

Dietary carotenoids, vitamin-A, vitamin-C, Dietary carotenoids, vitamin-A, vitamin-C, and vitamin-E, and advanced Age-related and vitamin-E, and advanced Age-related Macular DegenerationMacular Degeneration

Seddon et al.Seddon et al.J. Am. Med. Assoc. 272: (18) 1413-1420, 1994J. Am. Med. Assoc. 272: (18) 1413-1420, 1994

““Conclusion.-Increasing the consumption of foods rich in certain Conclusion.-Increasing the consumption of foods rich in certain carotenoids, in particular dark green, leafy vegetables, may decrease carotenoids, in particular dark green, leafy vegetables, may decrease the risk of developing advanced or exudative AMD, the most visually the risk of developing advanced or exudative AMD, the most visually disabling form of macular degeneration among older people.”disabling form of macular degeneration among older people.”

Lutein & Zeaxanthin in the MaculaLutein & Zeaxanthin in the Macula

• Macula is the Region Macula is the Region Directly Behind the Lens, Directly Behind the Lens, Receiving the Most Light.Receiving the Most Light.

• Lutein & Zeaxanthin are Lutein & Zeaxanthin are Deposited Specifically.Deposited Specifically.

• The Role Is To Prevent The Role Is To Prevent Photo-Oxidation. Photo-Oxidation. Macula

Lens

Lutein & Zeaxanthin

Epidemiological Studies Investigating Lutein and Cataract Risk

Study Comparison Outcome

spinach intake1 5 times/week vs. 1 time/month risk 39%carotenoid intake2 13.7 vs. 1.1 mg/day of lutein risk 22%carotenoid intake3 7.0 vs. 1.3 mg/day of lutein risk 19%antioxidant intake4 1.3 vs. 0.3 mg/day of lutein risk 50%serum carotenoids5 0.4 vs. 0.18 mol/L of lutein risk 30%

1Handelman, et al., 19882Chasan-Taber, et al., 19993Brown, et al., 19994Lyle, Mares-Perlman, Klein, Klein, Greger, 19995Lyle, Mares-Perlman, Klein, Klein, Palta, Bowen Greger, 1999

Vegetables, fruit, and cancer prevention: Vegetables, fruit, and cancer prevention: A reviewA reviewSteinmetz KA, Potter JDSteinmetz KA, Potter JD

Journal of the American Dietetic Association 96: (10) 1027-1039, 1996Journal of the American Dietetic Association 96: (10) 1027-1039, 1996

““The types of vegetables or fruit that most often appear to be The types of vegetables or fruit that most often appear to be protective against cancer are raw vegetables, followed by allium protective against cancer are raw vegetables, followed by allium vegetables, carrots, green vegetables, cruciferous vegetables, and vegetables, carrots, green vegetables, cruciferous vegetables, and tomatoes.” tomatoes.”

Bioavailability is the fraction of an ingested nutrient that is available for utilization in normal physiological functions, metabolism and/or storage.

In the context of this discussion, bioavailability of carotenoids is defined as the amount of these micronutrients that are absorbed by the intestinal enterocytes and transported in the bloodstream.

Absorption and Bioavailability

“Physical inaccessibility of carotenoids in plant tissues may reduce their bioavailability. It may be difficult to free beta-carotene in dark- green leafy vegetables from its matrix.

Our findings do not support the long-standing assumption that vitamin A deficiency can be combated by increasing the intake of dark-green leafy vegetables. “

Lack of Improvement in Vitamin A Status with Lack of Improvement in Vitamin A Status with Increased Consumption of Dark-Green Leafy Increased Consumption of Dark-Green Leafy

VegetablesVegetables

dee Pee, S., West, C.E., Muhilal, Karyadi, D. and Hautvast, J.G.A.J. Lancet 346: 75-81.

(1995)

Food Matrix of Spinach Is a Limiting Factor in Determining the Bioavailability of -Carotene and to a Lesser Extent of Lutein in Humans

(Castenmiller et al. J. Nutr. 129: 349-355, 1999)

controlcartenoid supplementwhole leaf spinach miced spinachliquefied spinachliquefied spinach plus fiber

Time (days)

-20 0 20 40

Ser

um to

tal

-c

arot

ene

( m

ol/L

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time (days)

-20 -10 0 10 20 30

Seru

m lu

tein

( m

ol/L

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Lutein 51%-Carotene

7%

CarotenoidsCarotenoids

Cleavage Cleavage

Oxidation Oxidation

Isomerization Isomerization

MetabolitesMetabolites

CarotenoidsCarotenoids LYMPH

PORTAL

MICELLESMICELLES

ChylomicronChylomicron

Carotenoid Absorption & MetabolismCarotenoid Absorption & Metabolism

Adapted from Erdman et al. Ann. N.Y. Acad. Sci. 691:76-85 (1993)

Plasma Plasma -Carotene Response after Meals Supplemented with -Carotene Response after Meals Supplemented with Dietary PectinDietary Pectin (Rock and Swendseid, Am. J. Clin Nutr. 1992)

Time (hours)

0 50 100 150 200

Pla

sm

a B

-ca

rote

ne

le

vels

(m

ol/L

)

0.0

0.5

1.0

1.5

2.0

2.5With pectinWithout pectin

Food Processing and Preparation Food Processing and Preparation

• • Physical and thermal treatment of foods causes Physical and thermal treatment of foods causes degradation of plant cell structural constituents.degradation of plant cell structural constituents.

• • Thermal processing brings about disruption of the Thermal processing brings about disruption of the carotenoid protein complexes.carotenoid protein complexes.

• • Inactivation of oxidizing enzymes results in less Inactivation of oxidizing enzymes results in less degradation and greater stability of carotenoids.degradation and greater stability of carotenoids.

• • These changes result in enhanced uptake and efficiency These changes result in enhanced uptake and efficiency of carotenoid absorption.of carotenoid absorption.

Per

cen

tage

Ch

ange

Per

cen

tage

Ch

ange

Altered “Bioavailability” By Food ProcessingAltered “Bioavailability” By Food Processing

All-transAll-trans

Rock, C., Lovalvo, J., Emenhiser, C., Ruffin, M., Flatt, S., Schwartz, S., Rock, C., Lovalvo, J., Emenhiser, C., Ruffin, M., Flatt, S., Schwartz, S., J. Nutr.,J. Nutr., 128: 913-916, 1998 128: 913-916, 1998..

Intake of carotenoids and retinol in Intake of carotenoids and retinol in relation to risk of prostate cancerrelation to risk of prostate cancer

Giovannucci et al.Giovannucci et al.

Journal of the National Cancer Institute, 87(23): 1767-76, 1995Journal of the National Cancer Institute, 87(23): 1767-76, 1995

““Combined intake of tomatoes, tomato sauce, and pizza (which Combined intake of tomatoes, tomato sauce, and pizza (which accounted for 82% of lycopene intake) was inversely associated accounted for 82% of lycopene intake) was inversely associated with risk of prostate cancer.”with risk of prostate cancer.”

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1R

elat

ive

Ris

k

<1 1 2-4 >5Servings per Week

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1R

elat

ive

Ris

k

<1 1 2-4 >5Servings per Week

R elative R isk of Prostate C ancer (N on A 1) and Intake of Tom ato Sauce in the H PFS.

R elative R isk of Prostate C ancer (N on A 1) and Intake of Tom ato Sauce in the H PFS.

T om ato Saucep, trend = 0.001T om ato Saucep, trend = 0.001

Epidemiological Data: Relative RisksEpidemiological Data: Relative Risks

TotalTotalOralOral

EsophagusEsophagus*Stomach*Stomach

ColonColonRectumRectum

PancreasPancreas*Lung*Lung

*Prostate*ProstateBladderBladder

BreastBreastCervixCervix

MesotheliomaMesothelioma Relative Risk: 0 0.5 1.0Relative Risk: 0 0.5 1.0 1.5 1.5

Statistically Significant

Not Statistically Significant

Giovannucci, 1999

Carotenoids as AntioxidantsCarotenoids as Antioxidants

Endogenous and ExogenousEndogenous and ExogenousReactive Oxygen Species (ROS)Reactive Oxygen Species (ROS)

AntioxidantsAntioxidants

Lipids, Proteins, DNALipids, Proteins, DNADamageDamage

Chronic DiseaseChronic Disease

Lycopene as a Singlet Oxygen QuencherLycopene as a Singlet Oxygen Quencher

• Lycopene is the most efficient singlet oxygen quencher and Lycopene is the most efficient singlet oxygen quencher and phenoxyl radical scavenger among the naturally occurring phenoxyl radical scavenger among the naturally occurring carotenoids.carotenoids.

Stahl and Sies, 1996. Lycopene: a biologically important carotenoid for Stahl and Sies, 1996. Lycopene: a biologically important carotenoid for humans? humans? Arch. Biochem. Biophys.Arch. Biochem. Biophys. 336:1-9. 336:1-9.

Mortensen and Skibsted, 1997. Importance of carotenoid structure in Mortensen and Skibsted, 1997. Importance of carotenoid structure in radical-scavenging reactions. radical-scavenging reactions. J. Agric. Food Chem.J. Agric. Food Chem. 45:2970-2977. 45:2970-2977.

Other Hypothesized Mechanism of ActionOther Hypothesized Mechanism of Action

Stimulate enzymes that detoxify toxins/carcinogensStimulate enzymes that detoxify toxins/carcinogens

Enhance DNA repair mechanismsEnhance DNA repair mechanisms

Increase apoptosis of cancer cellsIncrease apoptosis of cancer cells

Decrease cell proliferationDecrease cell proliferation

Possess anti-angiogenesis activityPossess anti-angiogenesis activity

Restore gap-junction communicationRestore gap-junction communication

PDA Chromatogram of Tomato Carotenoids

all-trans Lycopene

cis Lycopene

all-trans -Carotene

Selected Geometrical Isomers of LycopeneSelected Geometrical Isomers of Lycopene

all-all-trans trans lycopenelycopene

5-5-cis cis lycopenelycopene

9-9-cis cis lycopenelycopene

13-13-cis cis lycopenelycopene

7,9,9’,7’-7,9,9’,7’-ciscis lycopene lycopene

nm

250 300 350

400 450 500

360 (cis peak)

C. 464

A. 472

B. 435

A. All-trans lycopene

B. Tetra-cis lycopene

C. 13-cis lycopene

LycopeneLycopene

CC3030 Analysis of Lycopene Geometrical Isomers Analysis of Lycopene Geometrical Isomers

Am J Clin Nutr 1997; 66:116-22Am J Clin Nutr 1997; 66:116-22

Lycopene is more bioavailable from Lycopene is more bioavailable from tomato paste than from fresh tomatoestomato paste than from fresh tomatoesChristine Gärtner, Wilhem Stahl and Helmut SiesChristine Gärtner, Wilhem Stahl and Helmut Sies

ABSTRACTABSTRACT Lycopene bioavailability, from a single dose of fresh tomatoes or Lycopene bioavailability, from a single dose of fresh tomatoes or tomato paste (23 mg lycopene) ingested together with 15 g corn oil was compared by tomato paste (23 mg lycopene) ingested together with 15 g corn oil was compared by analyzing carotenoid concentrations in the chylomichron fraction. The lycopene isomer analyzing carotenoid concentrations in the chylomichron fraction. The lycopene isomer pattern was the same in both fresh tomatoes and tomato paste. The triacylglycerol pattern was the same in both fresh tomatoes and tomato paste. The triacylglycerol response in chylomicrons was not significantly different after both treatments. Ingestion response in chylomicrons was not significantly different after both treatments. Ingestion of tomato paste was found to yield 2.5-fold higher total and all-of tomato paste was found to yield 2.5-fold higher total and all-transtrans-lycopene peak -lycopene peak concentrations (P<0.05 and P<0.005, respectively) and 3.8-fold higher area under the concentrations (P<0.05 and P<0.005, respectively) and 3.8-fold higher area under the curve (AUC) responses (P<0.001) than ingestion of fresh tomatoes. The same was curve (AUC) responses (P<0.001) than ingestion of fresh tomatoes. The same was calculated for lycopene calculated for lycopene ciscis-isomers, but only the AUC response for the -isomers, but only the AUC response for the ciscis-isomers was -isomers was significantly higher than ingestion of tomato paste (P<0.005). No difference was observed significantly higher than ingestion of tomato paste (P<0.005). No difference was observed in the in the - and - and -carotene response. Thus, in humans, the bioavailability of lycopene is -carotene response. Thus, in humans, the bioavailability of lycopene is greater from tomato paste than from fresh tomatoes.greater from tomato paste than from fresh tomatoes.

Electron MicroscopyElectron Microscopy

Electron micrograph of lycopene crystalloids in mature red tomato chromoplast.

A. Lycopene crystalloids. B. Plastoglobulin-type sacs in which -carotene reportedly accumulates.

3.5 mm

B

A

Tomato Product Consumption – Tomato Product Consumption – Correlation with Plasma LycopeneCorrelation with Plasma Lycopene

Tomato SauceTomato Sauce p < 0.0001p < 0.0001

TomatoesTomatoes p < 0.056 p < 0.056

Lower Prostate Cancer Risk in Men with Elevated Plasma Lycopene Levels: Results of a Prospective Analysis (Gann et al., Cancer Research, 1999, 59: 1225-1230)

Commercial Products Study – Commercial Products Study – Experimental DesignExperimental Design

Vegetable JuiceVegetable Juice

Wash-out Meals Analysis( 14 days) (28 days)

Tomato SoupTomato Soup

36 subjects18 M, 18F

Tomato SauceTomato Sauce

Plasma Lycopene ResponsePlasma Lycopene Response

0.000

1.000

2.000

3.000

Basel

ine

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

um

ole

s/L

sauce soup Veg

washoutwashout

feedingfeeding

Allen et al. J. Nutr. 2003

Total Lycopene Levels in Buccal CellsTotal Lycopene Levels in Buccal Cells

0.0

1.5

3.0

4.5

0 1 2 3 4 5 6 7 8

JuiceSoupSauce

Week

Allen et al. J. Nutr. 2003

Time (minutes)

Ab

so

rban

ce

Pre-Treatment

Post-Treatment

0 2000

1.0

Conjugated Diene Formation

(nm

)

Lipoprotein Oxidation Lag Period (min, mean ± SE)Lipoprotein Oxidation Lag Period (min, mean ± SE)

CSCS

BaselineBaseline

64.7±2.464.7±2.4

TreatmentTreatment

70.1±4.070.1±4.0

SignificanceSignificance

p<0.05p<0.05

RTSRTS 64.7±2.464.7±2.4 68.3±2.468.3±2.4 p<0.05p<0.05

V8V8 64.7±2.464.7±2.4 71.7±4.071.7±4.0 p<0.01p<0.01

Two Stage In Vitro DigestionTwo Stage In Vitro Digestion

Garrett et al. (1999) J. Agric. Food Chem. 47:4301

Centrifugation

TestTestMealMeal

**

GastricGastricDigestaDigesta

GastricGastricPhasePhase

IntestinalIntestinalDigestaDigesta

IntestinalIntestinalPhasePhase *

AqueousMicellarFraction

*

Caco-2 Human Intestinal CellsCaco-2 Human Intestinal Cells

Caco-2 CellsTest Media

DMEM +

Micellar Fraction

Apical

Basolateral

Harvest CellsAnalyze for Pigments

Collect Basolateral MediaAnalyze for Pigments

4 hours 0-10 hours

Uptake Transport

Massive doses of a carotene-rich vegetable to the basic diet over a long period did not greatly increase serum carotene and vitamin A

levels and that most carotene was excreted.

Carotene Balances on Boys in Rwanda Where Carotene Balances on Boys in Rwanda Where Vitamin A Deficiency is PrevalentVitamin A Deficiency is Prevalent

Roels, Trout and Dujacquier. J. Nutr. 1958

Absorption of Absorption of ββ-Carotene and Vitamin A with -Carotene and Vitamin A with Different Levels of FatDifferent Levels of Fat (adapted from Roels et al. J. Nutr.

1958)

200 g carrots(n=5)

200 g carrots+ 20 ml olive oil (n=5)

day β-carotene(µg/100 ml

serum)

Vitamin A(µg/100 ml

serum)

β-carotene(µg/100 ml

serum)

Vitamin A(µg/100 ml

serum)

0 43.0 36.6 47.8 31.0

31 80.6 51.0 297.8 50.4

difference 37.6 14.4 250.0* 19.4

* significantly different from β-carotene serum levels without fat addition (unpaired Student t-test, p<0.005)

Addition of fats to the diet may contribute to the relief of vitamin A deficiency in this region.

Roels, Trout and Dujacquier. J. Nutr. 1958

CarotenoidsCarotenoids

Cleavage Cleavage

Oxidation Oxidation

Isomerization Isomerization

MetabolitesMetabolites

CarotenoidsCarotenoids LYMPH

PORTAL

MICELLESMICELLES

ChylomicronChylomicron

Carotenoid Absorption & MetabolismCarotenoid Absorption & Metabolism

Adapted from Erdman et al. Ann. N.Y. Acad. Sci. 691:76-85 (1993)

a

L

4 hour

L

6 hour

L

9 hour

b

a L

2 hourL

0 hour

Post-prandial Post-prandial ResponseResponse

440 mV Applied Potential20.0 nA Full Scale

Brown et al. Am. J. Clin Nutr. In press

(2004)

Carotenoid Response at Carotenoid Response at Three Different Lipid Three Different Lipid LevelsLevels

Fat Free 0 gFat Free 0 g Reduced Fat 6 gReduced Fat 6 g Full Fat 28 gFull Fat 28 g

Full Fat

Reduced Fat

Fat Free

Lettuce: 40g, Avocado: 75g, Carrots: 100g, Spinach:80 g

A) Salad

B) Salad with 150g of Avocado

C) Salad with 24g of Avocado Oil

obtained from the same subject at 5th hour

HPLC Chromatograms of TRL Fraction

after Consumption of;

10.0 15.0 20.0 25.0 30.0

0.0

5.0

10.0

15.0

20.0

R etention tim e (m inutes)

Re

sp

on

se

(n

A)

87654321

[500 m V ]

[440 m V ]

[380 m V ]

10.0 15.0 20.0 25.0 30.0

0 .0

5 .0

10.0

15.0

20.0

25.0

R etention tim e (m inu tes)

Re

sp

on

se

(n

A)

87654321

[500 m V]

[440 m V]

[380 m V]

10 .0 15 .0 20 .0 25 .0 30 .0

-5 .0

0 .0

5 .0

10 .0

15 .0

20 .0

25 .0

R eten tion tim e (m inu tes)

Re

sp

on

se

(n

A)

87654321

[500 m V]

[440 m V]

[380 m V]

lutein

β- carotene α-

carotene

Baseline Corrected Mean AUC Values for Each Treatment

-10

0

10

20

30

40

50

60

Salad Salad with 75 gavocado

Salad with 150 gavocado

Salad with 24 gavocado oil

nm

ol.

h/L

pla

sm

aalpha-carotene

beta-carotene

lutein

Values are based upon mean AUC ± SEM

Tomato VarietiesTomato Varieties

Postprandial Absorption of Tangerine vs High Lycopene (Pasta Sauce)

0

10

20

30

40

50

60

70

nm

ol.h

/L p

lasm

a

Tangerine PS Normal HLPS

Normalized AUC (Total Lycopene)

Accumulating evidence (epidemiological, cell-culture, animal and Accumulating evidence (epidemiological, cell-culture, animal and human) continues to associate health benefits with fruit and vegetable human) continues to associate health benefits with fruit and vegetable consumption.consumption.

Carotenoid pigments unique to fruits and vegetables, through Carotenoid pigments unique to fruits and vegetables, through their antioxidant activity, may be important components with their antioxidant activity, may be important components with beneficial physiological properties. beneficial physiological properties.

““Bioavailability” of carotenoids from specific food products is Bioavailability” of carotenoids from specific food products is influenced by a number of dietary factors including food processing influenced by a number of dietary factors including food processing treatments and product formulation (i.e. lipid content) and should be treatments and product formulation (i.e. lipid content) and should be determined to more accurately assess delivery of these phytonutrients determined to more accurately assess delivery of these phytonutrients from the diet. from the diet.

SummarySummary

Consumption of carotenoid containing F&V’s may have a Consumption of carotenoid containing F&V’s may have a protective effect against oxidative stress in vivo by decreasing protective effect against oxidative stress in vivo by decreasing the susceptibility of lipoproteins to oxidation. the susceptibility of lipoproteins to oxidation.

In vitro assays can be employed to mimic digestion and In vitro assays can be employed to mimic digestion and absorption processes resulting in estimates of in vivo absorption processes resulting in estimates of in vivo bioavailability for carotenoids.bioavailability for carotenoids.

Care must be taken not to make the assumption that all the Care must be taken not to make the assumption that all the health benefits brought about by fruit and vegetable health benefits brought about by fruit and vegetable consumption are attributed to a single class of compounds such consumption are attributed to a single class of compounds such as carotenoids.as carotenoids.

SummarySummary

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

• Steven K. Clinton, M.D., Ph.D.Steven K. Clinton, M.D., Ph.D. Internal Medicine, OSUInternal Medicine, OSU• Dave Francis, Ph.D.Dave Francis, Ph.D. Horticulture &Horticulture &

Crop Science, OSUCrop Science, OSU• Mark Failla, Ph.D.Mark Failla, Ph.D. Human Nutrition, OSUHuman Nutrition, OSU• Cheryl Rock, Ph.D.Cheryl Rock, Ph.D. UC - San DiegoUC - San Diego• Wendy White, Ph.D.Wendy White, Ph.D. Iowa State UniversityIowa State University

• Minhthy Nguyen, Ph.D.Minhthy Nguyen, Ph.D.• Charlotte M. Allen, Ph.D.Charlotte M. Allen, Ph.D.• Mario Ferruzzi, Ph.D.Mario Ferruzzi, Ph.D.• Nuray Unlu, Ph.D.Nuray Unlu, Ph.D.• Torsten Bohn, Ph.D.Torsten Bohn, Ph.D.• Craig Hadley, Ph.D.Craig Hadley, Ph.D.• Elena Vittadini, Ph.D.Elena Vittadini, Ph.D.

QuestionsQuestions