June 3 2009

Carotenoid bioavailability and consumption in

Luxembourg

Eric Biehler, Lucien Hoffmann and Torsten Bohn

OH

OH

luteinall-trans-lycopene

2nd OSQCAmeeting

June 3 2009

Overview

1. Introduction – Occurrence and Properties of Carotenoids

2. Carotenoids and Health Aspects

3. Carotenoid Absorption

4. Project CARO – Intake of Carotenoids

5. Outlook

June 3 2009

Group of compounds consisting of 40 C atoms/ 8 isoprene-units

- 600 known species (40 in diet of importance)

- without oxygen = carotenes

- containing oxygen = xantophylls → can form esters

Examples:

all-tr-lycopene

beta-carotene

lutein

OH

OH

1. Introduction – Importance of Carotenoids

June 3 2009

Cannot be synthesized by humans, but all plants, most bacteria and fungi

- Have to be consumed within the diet:

- Plant foods: fruits, vegetables

- Animal foods: eggs, some fish

- protection of photo-oxidation most important

- quenching effect/radical binding

1. Introduction – Importance of Carotenoids

Function of carotenoids in plants

Not entirely understood, but:

- increase the range of light absorption by chlorophylls

(closing the absorption gap between 450-500 nm)

June 3 2009

- Age related macular degeneration : lutein, zeaxanthin

protect tissue from degeneration due to oxidative reactions

(Landrum et al, 1997; Bernstein et al, 2001)

retina

- Pro-vitamin A activity: carotenoids can be transformed into retinol

Different efficiency: β-carotene (1/6), α-carotene (1/12), β -cryptoxanthin (1/12)

2. Carotenoids and Health - Vision

- Quenching effect of carotenoids, 1O2

- Direct reaction with radicals e.g. peroxyl radical O22-

protection from lipid peroxidation(within cell-membrane)

June 3 2009

Human studies (focus lycopene and beta-carotene):

2. Carotenoids and Health - CVD

Relation between:

Epidemiological studies: high intake or serum levels

Reduced markers of inflammation/atherosclerosis (Hozawa et al. 2007, CARDIO/Yalta study; Klipstein-Grobisch et al. 2000)

Supplementation/intervention studies:

Improved blood lipid markers (LDL) (Peto et al. 1995, similar Rao 1998)

- cholesterol synthesis inhibiting effect?- prevention of LDL from oxidation

Mechanism:

June 3 2009

Some evidence for healthy subjects, from whole foods

2. Carotenoids and Health - Cancer

- reduced risk of : prostate cancer digestive tract cancerslung-cancer

Higher lung cancer rate in smokers after β-carotene supplementation

acting as pro-oxidant if other synergist are missing (tocopherols, vit C ?)

- Finnish smoking study (CARET), - Australian ATBC, - VITAL Study (US)

Reduced serum levels (lycopene, total carotenoids)(Giovannuci 2005, Zhang 2007; Franceschi, 1994;

Gallicchio et al. 2009)

June 3 2009

2. Carotenoids and Health - Osteoporosis

Total carotenoids:

Some but limited evidence for post-menopausal women

- inhibit bone resorption, stimulate prolif. + diff. of osteoblasts- Oxidative stress ↓ → bone resorption ↓

Low BMD, especially insubjects at risk for osteoporosis

Reduced serum levels (total carotenoids) (Maggio et al. 2006, Sugiura et al. 2007, Wattanapenpaiboon 2003) (Sahni, 2009, FRAMINGHAM Osteoporosis Study)

June 3 2009

3. Carotenoid Absorption

Storage in tissue, e.g. liver

Free form or esterified

(xanthophylls)

Oral uptake

low absorption, via lymph,

1-10%feces

predominant excretion

(breakdown products)

Small losses (breakdown products)

urine, skin, sweat,hairs

www.colorado.edu

June 3 20092nd colloqueOSQCA

food pieces

carot.

carot.carot.

carot.carot.

carot.

oil droplets+

carotenoids

lipase etc. passiv, transcellular

passiv diffusion, facilitated

absorption limited,efflux transporters

lymph. vessel

enterocytes

intestinal lumen

carot.

carot.carot.

carot.carot.

carot.

carot.

carot.

carot.

carot.carot.

carot.carot.

carot.

carot.

carot.carot.

carot.carot.

carot.

mixed micelles

pancreatin

bile salts

→ as lipid soluble compounds, carotenoids have to be micellarised prior to absorption

bile salts

fatty acids and monoglycerides 25 nm

June 3 2009

S species of carotenoid

L molecular (chemical) linkage

A amount of carotenoids consumed

M food-matrix

E effectors of absorption and bioconversion

N nutrient status of the host

G genetic factors

H host-related factors such as GI passage

time, presence of infections

I interactions of the above factors

→ higher bioavailabilityprocessed foods, lipids

Bohn, Curr Nutr Fd Sci 2008

3. Carotenoid Absorption: Influence factors

esters: less polar → less well incorporated into micelle→ better protected in core of micelle ?

more bended structure → increased bioacessibility ?

xanthophylls preferrably micellarized

June 3 20092nd colloqueOSQCA

Group B (n=11)

Salad Salad + Salad + Salad + 24g avocado oil

100g shredded carrots: 6.2 mg a-carotene8.8 mg ß-carotene

80g baby spinach:3.6 mg ß-carotene 5.4 mg lutein

40g romaine lettuce0.41 mg ß-carotene0.44 mg lutein

75g ~half fruit 150g ~whole fruit

2 wk wo 2 wk wo 2 wk wo2 wk wo

Initiation of low-carotenoid diet

Salad + Salad + Salad + Salad + 24g avocado oil

100 g shredded carrots : 6.6 mg α-carotene7.5 mg β-carotene

80 g baby spinach:3.6 mg β-carotene 5.4 mg lutein

40 g romaine lettuce0.41 mg β-carotene 0.44 mg lutein

75 g ~ half fruit(12 g lipids)

150 g ~ whole fruit(24 g lipids)

2 wk wo 2 wk wo 2 wk wo2 wk wo

Initiation of low- carotenoid diet

Avocado study, n=11

3. Carotenoid Absorption: Influence factors (i.e.fat)

June 3 2009

Comparison of baseline corrected mean AUC values for each test meal

-1.32

***

**

*

*

*

-10

0

10

20

30

40

50

60

Salad Salad with 75g avocado

Salad with150 g

avocado

Salad with 24g avocado oil

nm

ol.h

/L p

lasm

a

alpha-carotene

beta-carotene

lutein

A A

A

B

ABB

B

BB

BB

B

3. Carotenoid Absorption: Influence factors (i.e.fat)

Unlu, Bohn et al., Jounal of Nutrition, 2005

- Presence of fat from avocado significantly increased carotenoid concentration in blood- no significant influence from the amount (12 vs. 24 g lipids) and/or the type of fat (avocado vs. oil)

June 3 2009

4. Project CARO – PART 1: carotenoid consumption

Carotenoid Analysis in Luxembourgish foods

food consumption pattern in Luxembourg

AIM (1) Carotenoid intake in Luxembourg

1

2

June 3 2009

4. Project CARO – PART 1: carotenoid consumption

- local food production (especially fruits, vegetables)

- consumption patterns inhomogeneous(divers Luxembourgish population, immigrant workers vs. natives)

- influence from different foreign cuisines

Why does Luxembourg need own data? Why not simply take data from neighbouring countries?

- no data on carotenoid consumption available- many food contents are not known

Food composition and consumption in Luxembourg

missing database: nutrients and non-nutrients

Limited use of data from other countries

June 3 2009

4. Project CARO – PART 1: carotenoid consumption

carotenoid analysis of frequently consumed fruits and vegetables (n=26)

Calculating the carotenoid uptake/day/capita

Combining disappearance data from the national statistical institute (STATEC) and a major local grocery chain (CACTUS S.A.)

+

June 3 2009

carrots (2.16 mg/d)

oranges (1.07 mg/d)

tomatoes (0.89 mg/d)

Major daily contributors

Contribution of daily carotenoid intake Comparison of carotenoid uptake:

(1) Eicholzer et al. 2005(2) DGE 2004(3) Elmadfa et al. 2004(4) O'Neill et al. 2001(5) Galan et al. 2003

GD Lux. 6.9mg/d/cFrance 16.1mg/d/cSwitzerland 4.2mg/d/c

4. Project CARO – PART 1: carotenoid consumption

June 3 2009

4. Project CARO – Part 2 (in vitro digestion)

impact on carotenoid absorption:interaction of macro- and

micronutrients

simulated digestion of foods: major carotenoid precursors

and degradation products

AIM (2) Carotenoid bioavailability

June 3 2009

stepwise adjustment to pH 7 with NaOH and NaCO3, 37 oCst

omac

h pH 2 with HCl, 37 oC, 1 hr, shaking bath, + pepsin + NaCl

pre-digested sample

smal

l in

test

ine Digesta

pH 7, 37 oC, 2 hrs shaking bath, + pancreatin + lipase + bile salts

sample adjusted to a final volume

aliquot for further examination

4. Project CARO – Part 2 (in vitro digestion)

*Based on the method of Miller et al. 1981 (AJCN)

“gastrointestinal passage”

Test meal + carotenoid containing fruit/vegetable

chew

ing

grinding + homogenization

In-vitro digestion of carotenoids*

June 3 2009

DigestaAliquot from the aqueous phase

Centrifugation at 100.000 g, 35 min

filtration 0.22 um

Quantification of carotenoid uptake

Estimation of carotenoid bioaccessibility

4. Project CARO – Part 2 (in vitro digestion)

Micellarization

June 3 2009

4. Project CARO – Part 2 - Preliminary Results

- Complex matrices/meals (fresubin, liver sausage) decrease carotenoid bioaccessibility

- the addition of fat seems to enhance bioaccessibility (skim milk<normal fat milk <high fat milk)

June 3 2009

4. Project CARO – Part 2 - Preliminary Results

- digestion without pepsin seems to slightly enhance carotenoid micellarization

- the loss of bile, pancreatin and total enzymes decrease carotenoid bioaccessibility

June 3 2009

5. Outlook:

II: Bioavailability: focus on bioavailability of individual carotenoidspecies, including a cell model, mimicking the small intestine

Ib. Intake : Study on consumers behaviour concrete study with actualpeople, including dietary recall

ORISCLAV-study of CRP-Santé (Alkerwi/Lair-Hillion)

Ia : Investigating additional carotenoid rich foods (juices, dairy products, sauces, convenient foods)

June 3 2009

This project is financed by the Fonds National de la Recherche Luxembourg (FNR).

We would like to thank CACTUS S.A. for the participation and support of the study.

Acknowledgements

June 3 2009

3. Carotenoid Absorption

1. Release from food matrix [mechanical, enzymatic]

2. Incorporation into mixed micelles [bile salts, lipids from foods]

3. Passive or active absorption [diffusion or SRB-1 uptake via protein carrier]

Aspects of release:

- chewing

- amylase

- acidity (stomach)

- proteinases

- lipase

Carotenoids are mostly bound to protein complexes, e.g. light harvestingprotein complexes in plant chloroplasts.

www.staff.uni-mainz.de/.../Bilder/PSI-modell.gif

June 3 2009

3. Carotenoid Absorption

→ as lipid soluble compounds, carotenoids have to be micellarised prior to absorption

bile salts

fatty acids and monoglycerides

Example of a mixed micelle in the small intestine:

25 nm

1. Release from food matrix [mechanical, enzymatic]

2. Incorporation into mixed micelles [bile salts, lipids from foods]

3. Passive or active absorption [diffusion or SRB-1 uptake via protein carrier]

June 3 2009

3. Carotenoid Absorption

1. Release from food matrix [mechanical, enzymatic]

2. Incorporation into mixed micelles [bile salts, lipids from foods]

3. Passive or active absorption [diffusion or SRB-1 uptake via protein carrier]