Selenium Biofortification and

Antioxidant Function:

Complexities and Interactions

John Finley

USDA-ARS

Se: A brief history

• Originally studied for

toxicity

• Shown to be essential in

1957

• Present DRI of 55ug/d

Se as an Antioxidant: A Rush to

Judgement?

• Exudative diathesis in chickens – indicative of tissue damage:

•Free Radicals?

Se as an Antioxidant

Glutathione Peroxidase: the first known Selenoprotein

Selenocysteine at active site

Reaction:

2GSH + H2O2 → GS–SG + 2H2O

Se as an Antioxidant

Reaction of GPX fit within hypothesized scheme of antioxidant

protection

Se as an Antioxidant

• Selenium deficiency = signs of oxidative stress

• First selenoprotein = activity to reduce hydrogen peroxides

• Subsequent selenoproteins = have same reactive site:

(selenocysteine)

Selenium = Powerful Antioxidant

Need to fortify low and deficient popuulations

Selenium: A trace mineral with

antioxidant properties, selenium may

be useful in preventing arthritis and

other conditions, including age-related

blindness, cancers, cardiovascular

disease, cataracts and kidney

disease. You’ll get selenium from

whole-grain wheat products and

shellfish, such as oysters and crab. A recent study raised the

possibility that taking selenium supplements might

increase the risk of developing diabetes, so talk to

your doctor before taking extra selenium. Getting

more selenium in your diet appears safe.

Selenium as an Antioxidant:

Current Thinking

• Other ideas as to GPX function:• Cell signaling

• Se storage protein

• No protein positively associated with Se-

deficiency diseases

• No positive health benefits in Westernized

nations that have fortified

Selenium BioFortifcation:

Complexities

Selenium variation in

foods from different

geographical

areas and/or different

suppliers (Finley et

al. 1996)

Food Se (mg/100g)Food Se (mg/100g)

Corn masa mix, Maseca 4.7±0.6

Corn masa mix, Masa Harina 49.8±0.8

Flour white all-purpose, Gold Medal 15.9±0.9

Flour, white all-purpose, Pillsbury 39.9±1.3

Ground beef - North Dakota 33.6±0.7

Ground beef - SW Missouri 6.0±1.2

Selenium in pasta

USDA Nutrient Database

Food Selenium/serving

Spaghetti 20.2 µg Se

Whole-wheat spaghetti 23.7 µg Se

Macaroni 20.2 µg

Average selenium content of pasta analyzed

at Grand Forks Human Nutrition Center

20.5 µg/serving

Selenium in pasta

Italian pasta

Brand ug Se/100g

Venecia, angel hair 6.7±0.1

Davinci, fusilli 7.1±0.2

Venecia, linguini 5.7±1.0

Avg Italian brands 6.2

American pasta

Brand ug Se/100g

Market Pantry, spaghetti 139±7.0

Creamette, shells 102±11

Leonardo, shells 100±10

Avg American brands 57.4

Se Distribution in U.S. Soils

= Low Se

::::::::

:::: = Variable Se

::::::::

::::

…= Se toxicity

Kubota and

Allaway, 1972

Volcanic soils, low pH = Se deficient/low bioavailability

Jurrasic Shale = high Se

Weathering = Se released to soil

Meeting Se DRI with Se-enriched

Beef Amount of Se supplied by a 100g serving of

beef

0

50

100

150

200

250

Se,

ug

Men Women U.S. avg ND grass-fed SD feedlot-fed

DRI 100 g Beef

Se Accumulation in Plants

• Wheat = Accumulates Se in direct relationship

to amount in soil

•Total conc. 0.01 -15 pp

• Se Accumulator = Accumulates Se well

beyond availability from soil

• Broccoli = up to 1500 ppm;

• Astragalus = up to 10,000 ppm

NH3

COOH

WHEAT

CH-CH2-CH2-Se-CH3Selenomethionine

Transulfuration

pathway

NH3

COOH

CH-CH2-SeH

Selenocysteine

General proteinsLyase

SeH2SeO4SeO3 GS-Se-SG

Lyase

GSH GSH

NH3

COOHCH-CH2-Se-CH3

Se-methyl-selenocysteine

H-Se-CH3

Lyase

CH3-Se-CH3 BREATH

tRNA-C-C-COH

O tRNA-serine

tRNA-C-C-C-SeH

O

Lyase

CH3-Se-CH3

tRNA-selenocystein

Selenoprotein incorporation

CH3URINE

MEAT

MEAT

BROCCOLI

GARLIC

SELENATE SELENITE

GSH-Px (5)deIodinase (3)Selenoprotein pSelenoprotein WTRSelenophosphate synthase 215kDa selenoprotein18kDa selenoprotein

Form Matters – understand the chemistry

Selenium from broccoli does not activate rat

TR mRNA (A) or accumulate in muscle (B)

as readily as other food forms of Se.

0.0

0.5

1.0

1.5

0 0.5 1 1.5 2

Dietary Selenium (ug/g)

C

0 0.5 1 1.5 2

C1

C2

C3

C4

1

1.5

2

2.5

3

3.5

4

0 0.5 1 1.5 2

Dietary Selenium (ug/g)

BA

250 kDa

98 kDa 64 kDa

50 kDa

30 kDa

16 kDa

6 kDa

MW Meat Broc Meat BrocMeat Broc Meat BrocLiver Kidney Testis Plasma

Incorporation of 75Se from broccoli

or meat into selenoproteins

Selenium from meat incorporates into

selenoproteins

But Se from broccoli does not

Selenium from

broccoli does not

accumulate in the

plasma of healthy

men as well as Se

salts

Form matters – but does it in

real life?

• Western diet = mixed foods, mixed origin

Selenium BioFortification:

Interactions

Broccoli Extract SF

(mM)

Se

(mM)

0 SM 239 2 c 1.3 0.2 a

100 SM 157 3 b 35.4 1.3 b

1000 SM 41 3 a 354.0 5.6 c

Selenium fertilization decreases

sulforaphane content of broccoliRobbins, Banuelos, Finley and Keck, 2004

Selenium fertilization alters

phenolic acid profile in broccoli

0 ppm Se

1000 ppm Se

1,2'-disinapoyl-2-

feruloyl gentiobiose

(11), 1-sinapoyl-2-

feruloyl gentiobiose

(8), 1,2,2'-trisinapoyl

gentiobiose (10) and

1,2-disinapoyl

gentiobiose (7), 1,2-

diferuloylgentiobiose

(9), and 3-O-caffeoyl-

quinic (neochlorogenic

acid) (2).

0

5

10

15

20

TR

Ac

tiv

ity

mU

/mg

pro

t/m

in

* *

TRT 6

Cntrl 20% broc 3.2 umol Sf/d 52 umol Sf/d 20% B +1.6 Sf 20% B + 50 Sf

Thioredoxin reductase in livers of rats fed broccoli or SF

0

100

200

300

400

500

600

700

800

900

GS

H-P

x A

ctiv

ity

mU

/mg

pro

t/m

in

**

Cntrl 20% broc 3.2 umol Sf/d 52 umol Sf/d 20% B +1.6 Sf 20% B + 50 Sf

Glutathione peroxidase in livers of rats fed broccoli or SF

1 2 3 4 5 6 7 8 9 10 11 12

Lanes 1-2- AIN 76 Diet

Lanes 3-4- 20% Broccoli Diet

Lanes 5-6- 0.32 mMol SF/kg diet

Lanes 7-8 5.16 mMol SF/kg diet

Lanes 9-10 20% Broccoli Diet + 0.16 mMol SF/kg diet

Lanes 11-12 20% Broccoli Diet + 5 mMol SF/kg diet

GSH-Px

TR

Effect of Sulforaphane from broccoli on Rat

Hepatic GSH-Px and TR Protein

Concerted regulation of multiple

Antioxidant proteins

Consensus ARE Sequence (GST-A2)

5’-GTGACAAAGCA-3’

(-57) 5’-ATGACAAAGCA-3’(-46)

TR Promoter

Putative Antioxidant Response Element (ARE)

in TR Promoter

Effect of Sulforaphane on TR or QR

Transcription (luciferase promoter

construct)

0

10

20

30

40

50

Fir

efl

y/r

en

illa

rati

o5.0 uM SF

2.0 uM SF

0.5 uM SF

0 uM SF

TR

Effect of Sulforaphane on TR or QR

Transcription (luciferase promoter

construct)

0

2

4

6

8

10

12

14

16

fire

fly

/ren

illa

rati

o

5.0 uM SF

2.0 uM SF

0.5 uM SF

0 uM SF

0

10

20

30

40

50

Fir

efl

y/r

en

illa

rati

o5.0 uM SF

2.0 uM SF

0.5 uM SF

0 uM SF

TR

QR

Broccoli Extract SF

(mM)

Se

(mM)

0 SM 239 2 c 1.3 0.2 a

100 SM 157 3 b 35.4 1.3 b

1000 SM 41 3 a 354.0 5.6 c

Selenium fertilization decreases

sulforaphane content of broccoliRobbins, Banuelos, Finley and Keck, 2004

ANOVA

Treatment P value

Effect of selenite 0.0009

Effect of SF 0.29

Interaction Se*SF 0.33

0

20

40

60

80

100

120G

SH

-Px

Act

ivit

y

mU

/mg

pro

t./m

in

C SF Se SF+Se

GPx activity in Hepa 1c1c7 cells

Se and SF synergistically

upregulate thioredoxin reductase

activity

0

10

20

30

40

50

60T

R A

cti

vit

y (

mU

/mg

pro

t./m

in)

A

B B

C

Control 2uM Sel 2uM SF 2uM Se and

2uM SF

ANOVA

Treatment P value

Effect of selenite 0.69

Effect of SF <0.0001

Interaction Se*SF 0.14

0

100

200

300

400

500

600

QR

Act

ivity

nmol

DPI

P/m

g pr

ot./m

in

C SF Se SF+Se

Quinone reductase in Hepa 1c1c7 cells

Se fertilization affects the

ability of broccoli

extracts to induce TR and

QR activity

Summary

• TR is transcriptionally regulated by an ARE

• Transcriptional regulation increases TR activity beyond the max induced by Se

• Sf from broccoli regulates TR activity

• Sf + Se = additive induction of TR

• Se antagonizes Sf accumulation in broccoli and vice versa

Summary – Lessons learned

1. The real story is often more complex than

the theory

Summary – Lessons learned

1. The real story is often more complex than

the theory

2. Understanding nutritional chemistry is

essential

Summary – Lessons learned

1. The real story is often more complex than

the theory

2. Understanding nutritional chemistry is

essential

3. Understanding the soil/plant interaction is

essential

Summary – Lessons learned

1. The real story is often more complex than

the theory

2. Understanding nutritional chemistry is

essential

3. Understanding the soil/plant interaction is

essential

4. Enrichment of one nutrient may result in

unexpected interactions, both in the plant

and the animal that consumes it

Collaborators and partners

• Cindy Davis, NCI

• Joel Caton, NDSU

• Mark Roberge, UND

• Korry Hintze, NDSU

• Anna Keck, U. Ill.

• Allan Hovland, SD

• Phil Whanger, OSU

• Gary Banuelos,

USDA

• Mike Grusak, USDA

• Karl Wald, GFHNRC

• Eliz. Jeffery, U. Ill.

• Yi Feng, U. Louis.