Download - CAROTENOIDS, INFLAMMATION AND ASTHMA
CAROTENOIDS, INFLAMMATION AND ASTHMA
Dr Lisa WoodDr Lisa Wood
Centre for Asthma and Respiratory Diseases
Hunter Medical Research Institute
University of Newcastle
CAROTENOIDS –IMPORTANT DIETARY
ANTIOXIDANTSANTIOXIDANTS
ANTIOXIDANTS
ANTIOXIDANTS protect the body from the damaging effects of free radicals, preventing OXIDATIVE STRESS. Mechanisms include: scavenging of free radicals, binding of metal ions, regeneration of other AO.
3
ENDOGENOUS
ANTIOXIDANTS
eg Glutathione
GSHPx
SOD
Catalase
DIETARY
ANTIOXIDANTS
eg Vitamin C
Vitamin E
Flavonoids
Carotenoids
CAROTENOIDS
• Natural fat-soluble pigments found mainly in plants
• Red, orange, yellow pigments
• Most prevalent as trans-isomers
• Interact with lipophilic cell components eg membranes and lipid globules
• Sensitive to heat and light
• Humans cannot synthesize
• 600 carotenoids isolated from natural sources– 40 found in Western diet
– 21 found in human plasma
α-carotene: β-carotene:
Lutein:
STRUCTURE
Lycopene: Zeaxanthin:
Carotenoids
Carotenes Xanthophylls
α-carotene
β-carotene
Lutein
Zeaxanthin
CLASSIFICATION
Lycopene Cryptoxanthin
ANTIOXIDANT PROPERTIES OF CAROTENOIDS
• Singlet oxygen quenching
• Free radical quenching
– CAR + ROO• → CAR •+ + ROO- (Electron transfer)
– CAR + ROO• → CAR • + ROOH (Hydrogen abstraction)– CAR + ROO• → CAR • + ROOH (Hydrogen abstraction)
– CAR + ROO• → ROOCAR• (Addition)
• Increased activity of antioxidant enzymes
• Lycopene > α-carotene > β-cryptoxanthin
> zeaxanthin= β -carotene > lutein
FOOD SOURCESFood ββββ-carotene (µg/100
g):αααα-carotene Lutein+Zeaxanthin Lycopene
Apricot 3524 0 0 5
Broccoli 700 1 1900 0
Carrot 7900 3600 260 0
Mango 1300 0 0 0
Nectarine 103 0 NA NA
Peach 99 1 14 0
Pumpkin 3100 3800 1500 0
Spinach 4100 0 10200 0
Tomato paste 1700 NA NA 6500
Tomato juice 900 NA NA 8500
Tomato raw 520 NA 100 3100
Watermelon 230 1 14 4100
(Mahan, 2004)
BIOAVAILABILITY
Fat essential for lycopene uptake:
Subjects had ingested a fresh vegetable salad with salad dressing containing:▼ 0g(fat-free),
○ 6g(reducedfat), or
● 28g (full-fat) canola oil; n=7.
(Brown, AJCN, 2004)
ASTHMA –A CHRONIC INFLAMMATORY DISEASE OF THE AIRWAYS
ASTHMA PREVALENCE
� Prevalence: increased over time, higher in Westernised countries � Migration from developing to Western country can lead to asthma (Gibson, 03)
ENVIRONMENTAL FACTORS INFLUENCING
DEVELOPMENT AND EXPRESSION OF ASTHMA
Allergens: Indoor (dust mites, dogs, cats, cockroaches)Outdoors (pollens, molds, fungi, yeasts)
Infections (early childhood)Occupational exposureSmoking (active/ passive)
Diet = Antioxidants, Dietary Fat, Obesity
(GINA, 2004)
ANTIOXIDANTS AND ASTHMA
� Oxidative stress occurs in asthma because..asthmatics exhibit an exaggerated immune response to stimuli (e.g. allergens and viruses). Airway inflammatory cells respond with a ‘respiratory burst’, involving oxygen cells respond with a ‘respiratory burst’, involving oxygen uptake and leakage of free radicals into surrounding cells.
INFLAMMATORY CELL ACTIVATION
ACQUIRED IMMUNITY INNATE IMMUNITY
AllergenNO2/Ozone Chemicals
Activated TH2 cells
IL-5
Eosinophils
IL-8
Neutrophils
Activated epithelial cells/macrophages
Virus Endotoxin
MECHANISMS LEADING TO OXIDATIVE STRESS IN ASTHMA
OXIDATIVE STRESS
ASTHMA
Airway Hyperresponsiveness, Smooth Muscle Contraction, Mucus Hypersecretion, Epithelial Cell Shedding
Mediator Release including ROS (O2.-, H2O2,
.OH)
Eosinophils Neutrophils
2
(Wood et al; ERJ, 2003)
N
EFFECTS OF OXIDATIVE STRESS IN ASTHMA
Air enters respiratory tract via mouth
and nose and enters bronchial tubes
ASTHMA:
Inflamed bronchial tube NORMAL
bronchial tube
Smooth muscle contracts and becomes ‘twitchy’
Excess mucus
500
1000ta
ne (
pg/m
L)
OXIDATIVE STRESS IN ASTHMA
Controls
Healthy
Asthma
Stable Asthma
Post Exac
0
8-isopro
st
Exac
Asthma
(Wood, AJRCCM, 2005)
INFLAMMATORY CELL ACTIVATION
ACQUIRED IMMUNITY INNATE IMMUNITY
AllergenNO2/Ozone Chemicals
Activated TH2 cells
IL-5
Eosinophils
IL-8
Neutrophils
Activated epithelial cells/macrophages
Virus Endotoxin
MECHANISMS LEADING TO OXIDATIVE STRESS IN ASTHMA
OXIDATIVE STRESS
ASTHMA
Airway Hyperresponsiveness, Smooth Muscle Contraction, Mucus Hypersecretion, Epithelial Cell Shedding
Mediator Release including ROS (O2.-, H2O2,
.OH)
Eosinophils Neutrophils
2
(Wood et al; ERJ, 2003)
Antioxidant supplementation
quercetincatechinsphloridzin
Cough, wheeze (Tabak, 2001)Asthma (Shaheen,2001)FEV1 (Butland, 1999)
Apples/pears
vitamin Ccarotenoidsflavonoids
Wheeze (Butland, 99)FEV1 (Cook,97; Carey,98)Chronic lung disease (Miedema,93)
Fresh fruit
Active AO?Respiratory MarkerAO-rich Food
quercetincatechinsphloridzin
Cough, wheeze (Tabak, 2001)Asthma (Shaheen,2001)FEV1 (Butland, 1999)
Apples/pears
vitamin Ccarotenoidsflavonoids
Wheeze (Butland, 99)FEV1 (Cook,97; Carey,98)Chronic lung disease (Miedema,93)
Fresh fruit
Active AO?Respiratory MarkerAO-rich Food
EPIDEMIOLOGY OF ANTIOXIDANT-RICH FOODS AND ASTHMA
carotenoidsflavonoids
Wheeze (Ellwood,01; Hijazi,00)Bronchitis/asthma (LaVecchia,97)
Vegetables and Vegetable prod
lycopenequercetin
Asthma onset (Troisi, 1995)Tomato prod (pste/sce/pizza)
Vit E, SeWheeze (Ellwood, 01)Cereals
anthocyaninquercetin
Asthma severity (Shaheen, 01)Red Wine
carotenoidsflavonoids
Wheeze (Ellwood,01; Hijazi,00)Bronchitis/asthma (LaVecchia,97)
Vegetables and Vegetable prod
lycopenequercetin
Asthma onset (Troisi, 1995)Tomato prod (pste/sce/pizza)
Vit E, SeWheeze (Ellwood, 01)Cereals
anthocyaninquercetin
Asthma severity (Shaheen, 01)Red Wine
It is probable that a combination of nutrients are
providing protective effect
PLASMA CAROTENOID LEVELS IN ASTHMA
400500600700800900
100011001200
200
300
400
500(υg/L) (µg/L)
** * *
Asthma
Controls
0100200300400
0
100
**
* **
*
Total Carotenoids
lutein β-crypto-xanthin
lycopene α-carotene β-carotene
Plasma carotenoids reduced in asthma despite no differences in dietary intake
(Wood et al; JACN, 2005)
INVESTIGATINGLYCOPENE SUPPLEMENTATION TO PROTECT AGAINST ASTHMA
TRIGGERSTRIGGERS
VIRUSES ALLERGY
TRIGGER: Allergy
day -14 0 12 13 14 15 16
ip OVA sacrificeCommence daily nutrient
supplementation in OVA
Mice were sensitized by
intraperitoneal (ip) injection of 50 µg of OVA
Mice were challenged by intranasal (in) instillation of 10 µg of OVA
naive PBS OVA-con OVA-lyc0
1
2
3
4
5
6
7
8
9
*
* *
eo
sin
op
hil
s/m
L B
AL
F (
x10
4)
TRIGGER: Allergy
naive PBS OVA-con OVA-lyc0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
*
* *
blo
od
eo
sin
op
hil
s (
%)
40
naive PBS OVA-con OVA-lyc0
5
10
15
20
*
* *eo
sin
op
hil
s/1
00
µµ µµM
air
way
naive PBS OVA-con OVA-lyc0
10
20
30
40
*
* *
MS
C/1
00
µµ µµM
air
way
(Hazelwood et al, submitted July 2009)
Supplementation reduced eosinophilic infiltrates in the bronchoalveolar lavage fluid, lung tissue and blood, and mucus secreting cell numbers in the airways.
0.1
0.2
0.3
*
* *
IL-4
(n
g/m
L)
20
30
40
*IL-5
(n
g/m
L)
TRIGGER: Allergynaive
PBS
OVA-con
OVA-lyc
unstimulated OVA-stimulated0.0
0.1 *
IL-4
(n
g/m
L)
unstimulated OVA-stimulated0
10*
* *
IL-5
(n
g/m
L)
(Hazelwood et al, submitted July 2009)
Supplementation reduced ovalbumin-specific release of Th2-associated cytokines IL-4 and IL-5 was also reduced.
100
150
200
250 P = 0.026P = 0.002
IL-6
concentr
ation (pg/m
l)
1 0 0
1 5 0
2 0 0
2 5 0 p = 0 .0 0 2 p = 0 .0 0 1
(a)
IP-1
0 c
oncentration (pg/m
l)I
TRIGGER: Virus Infection
Cel
l alo
ne
Cel
l + R
V-1B
Cel
l+0.
5% T
HF +
RV-1
B
Cel
l+0.
5% T
HF/L
ycopen
e + R
V-1B
0
50
IL-6
concentr
ation (pg/m
l)
Cel
l alo
ne
Cel
l + R
V43
Cel
l+0.
5% T
HF +
RV43
Cel
l+0.
5%TH
F/Lyc
opene+
RV43
0
5 0
IP-1
0 c
oncentration (pg/m
l)I
(b )(Saedisomeolia, J Nutr Biochem, 2009)
Pre-incubation of airway epithelial cells with lycopene (dissolved in THF):- 24% reduction in IL-6 after rhinovirus-1B infection- 31% reduction in IP-10 after rhinovirus-43 infection
500000
1000000
1500000
2000000
2500000
p =0.18
p = 0.025
TC
ID50 /
ml
TRIGGER: Virus Infection
Cel
l alo
ne
Cel
l +TH
F/lyco
pene
Cel
ls +
RV43
Cel
ls +
RV43
+ T
HF/ly
copen
eC
ells
+ R
V1B
Cel
ls +
RV1B
+ T
HF/ly
copen
e
0
(Saedisomeolia, J Nutr Biochem, 2009)
Pre-incubation of airway epithelial cells with lycopene (dissolved in THF):- 85% reduction in rhinovirus-1B replication.
INVESTIGATINGMANIPULATION OF DIETARY
ANTIOXIDANT INTAKE IN ANTIOXIDANT INTAKE IN ASTHMA
STUDY DESIGN: Subjects advised to:
1. Limit Vegetable/salad intake - 2 serves per day 2. Limit Fruit (fresh, dried, tinned) intake - 1 serve/day
3. Replace whole grain breads with processed products4. Avoid antioxidant additives (300 – 322)
5. Avoid high antioxidant foods eg. nuts, tea, red wine
DIETARY ANTIOXIDANT DEPLETION
1. Carotenoid levels reduced
00.20.40.6
0.81
1.2
*
*** *
*p<0.05
0
Total
Car
oten
oids
Lute
in/ Z
eaxa
nthi
n
beta
-cry
ptox
anth
in
Lyco
pene
alph
a-ca
rote
nebe
ta-c
arote
ne
Baseline Follow ing 10 days on Low Antioxidant Diet
*
2. Airway inflammation worsened
90
80
70
60
50
%S
pu
tum
Ne
utr
op
hils
P=0.038
Low AO Diet
50
40
30
20
10
0
Baseline
%S
pu
tum
Ne
utr
op
hils
(Wood, Free Rad Res, 2008)
95
100
105
110
Baseline Low AO Diet
1
1.2
1.4
1.6
1.8
3. Asthma worsened%
pre
dic
ted
Lu
ng
Fu
nctio
n
Asth
ma
Co
ntr
ol S
co
re
P=0.004
P=0.002
P=0.035
60
65
70
75
80
85
90
95
0
0.2
0.4
0.6
0.8
1
% p
red
icte
d L
un
g F
un
ctio
n
%FEV1 %FVC
Asth
ma
Co
ntr
ol S
co
re
(Wood, Free Rad Res, 2008)
GENOMICS OF ANTIOXIDANT WITHDRAWAL IN ASTHMA
Gene profiling of sputum samples following dietary antioxidant withdrawal
104 genes differentially expressed 22 up, 82 down.
Upregulated genes:Innate Immune Receptors (TLR2, IL1R2, CD93, ANTXR2)
Signalling Molecules (IRAK2, IRAK3, MAP3K8)
Proteases (MMP25, CPD)
Regulation of Apoptosis (CFLAR)
Visit 1 (red) V2 (blue)
(Baines, OMICS, 2009)
• Downregulated genes:– Glutathione metabolism (GSTA1, GSTA2)
– Protease Inhibitors (SLPI, SERPINB3)
– Eosinophil and Mast Cell Responses (CLC, TPSAB1)
• 5 genes including IRAK2, IL1R2, TLR2, SERPINB3, C20orf114 confirmed to be differentially expressed by real time PCRreal time PCR
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4
Microarray
qPCR
Fold Change from Baseline
TLR2
IL1R2
IRAK2
C20orf114
SERPINB3
(Baines, OMICS, 2009)
Plasma Antioxidants vs airway IRAK3 Gene Expression
IRAK3 gene expression is:
- Correlated with Total plasma antioxidants
- Increased by antioxidant withdrawal
- Increased in sputum in
13
14
15
To
tal A
ntio
xid
an
ts
- Increased in sputum in neutrophilic asthma
- Reported to be involved in tolerance to LPS and immunosuppression in sepsis
- Reported to be associated with early onset persistent asthma
11
12
To
tal A
ntio
xid
an
ts
-1 -.5 0 .5 1IRAK3
r=0.83
p=0.0029
(Baines, OMICS, 2009)
Wash
out
Treatment 1
Wash out
Treatment 2
Washout
Treatment 3
STUDY DESIGN:
LYCOPENE SUPPLEMENTATION IN STABLE ASTHMA
Visit 1 2 3 4 5 6 7
VS VS PLACEBO
Placebo Juice Extract0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
p<0.01
p<0.001
Ly
co
pe
ne
( u
g/m
L)
Placebo Juice Extract0
25
50
75
100
p<0.05
p<0.05
Neu
tro
ph
il E
lasta
se A
cti
vit
y (
ng
/mL
)%
Neu
tro
ph
ils
� Increased plasma lycopene
� Reduced airway neutrophils
� Reduced airway NE activity
Placebo Juice Extract0
1000
2000
3000
p<0.05
45008500
12500
Neu
tro
ph
il E
lasta
se A
cti
vit
y (
ng
/mL
)
(Wood, Free Rad Res, 2008)
-20
-10
0n F
EV
1
xerc
ise
*
STUDY DESIGN: 30 mg /day lycopene for 7 days, n = 20
LYCOPENE SUPPLEMENTATION IN EXERCISE-INDUCED ASTHMA
Before
Treatment
Follow ing
Treatment
-50
-40
-30
-20
% C
hange in
follo
win
g e
x
* p<0.05(Neuman, Allergy, 2000)
CONCLUSIONS AND FUTURE DIRECTIONS
• Low intake of antioxidant-rich foods, such as fruit and vegetables, may be contributing to increased asthma prevalence.asthma prevalence.
• Antioxidant supplementation, in particular lycopene supplementation may be useful in asthma.
• Large scale trials are needed to establish the clinical efficacy of lycopene supplementation in asthma.
ACKNOWLEDGEMENTS
• Prof Peter Gibson• Prof Manohar Garg• Prof Paul Foster• A/Prof Phil Hansbro• A/Prof Peter Wark• Dr Katie Baines• Dr Katie Baines• Dr Ahmad Saedisomeolia• Leia Hazelwood• Heather Powell
• Centre for Asthma and Respiratory Diseases, Clinical and Laboratory Teams, HMRI, John Hunter Hospital and David Maddison Building, University of Newcastle