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Identify plasma metabolites altered in humans following sulforaphane consumption using novel, unbiased metabolomic technology.

Understanding mechanisms responsible for SFN’s health benefits is critical for developing dietary strategies using SFN to promote health and prevent disease. This work will provide information for directing further human studies to understand mechanisms by which SFN and cruciferous vegetable consumption promote health.

The metabolome of plasma samples from each time point was analyzed by HPLC tandem mass spectrometry.

Plasma Processing for Metabolomic Analysis:• Plasma collected from Histopaque separation procedure7 &

stored at -80oC. • Metabolites extracted from plasma by addition of 150 µl ice-cold

50:50 (v:v) methanol:ethanol.• Samples centrifuged for 15 minutes & stored at -80oC until

analysis.• Quality control (QC) samples prepared by combining 15 µl of

each sample into a single vial.

Metabolomic Analysis:• High-performance liquid chromatography (HPLC)

information/settings:• HPLC Column: Inertsil ® Phenyl-3 5 µM, 4.6 x 150 mm

(GL Sciences, Inc., Tokyo, Japan) • Cooling temperature: 15oC • Solvents: Methanol + 0.1% (v/v) formic acid (FA)

(organic), water + 0.1% (v/v) FA (aqueous)• Flow rate: 0.4 ml/min• Injection volume: 10 µl plasma

40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130m/z, Da

0100200300400500600700800900

10001100120013001400150016001700180019002000210022002299

Intensity, cps

44.0494

43.0289

114.0664

44.2680 86.071072.0439114.757255.028457.0429

I. Introduction

Plasma metabolites altered by sulforaphane in humansKim Kelsey1, Lauren L. Atwell1,2, John D. Clarke1, Anna Hsu1, Deborah Bella1, Jan F. Stevens2,3, Roderick H. Dashwood2,4, David E. Williams2,4, and Emily Ho1,2

1School of Biological and Population Health Sciences, 2Linus Pauling Institute, 3Department of Pharmaceutical Sciences, 4Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331

COLLEGE OF PUBLIC HEALTH AND HUMAN SCIENCES

Head 3 Head 3, to label the table below

Figure 1. Formation and metabolism of SFN. (A) Hydrolysis of GFN to SFN via myrosinase. (B) Metabolism of SFN via the mercapturic acid pathway. GFN, glucoraphanin; SFN, sulforaphane; GST, Glutathione-S-transferase; GTP, γ-Glutamyltranspeptidase; CGase, Cysteinylglycinase; NAT, N-acetyltransferase.

B)

Figure 2. Human study timeline. Healthy adults (n=10), ages 20-60 years, consumed a single dose of 200 µmol SFN equivalents from fresh broccoli sprouts. Subjects provided blood samples at time 0, 3, 6, 12, 24, & 48 hours after sprout consumption to determine what changes occur early and what changes occur later. = time of sprout consumption

*Blood draw

0 h 3 h 6 h 12 h 24 h

Blood draw

48 h-20 -15 -10 -5 0 5 10 15 20

D1 Score

-20

-15

-10

-5

0

5

10

15

20

25

D2

Sco

re

604 24 -2

604 12

604 0 774 0

604 48 676 12

788 0714 3

723 12 788 12

751 0618 0

724 12

604 3

604 24

723 0 676 0

714 12618 12714 24 723 6724 24

724 0 774 3 676 3

714 0

676 6604 12 -2

790 3 723 48

723 24

724 48

618 48

714 48

790 48618 3

774 12

724 3

788 6

774 6

604 6

790 12

790 0

790 24

751 6 788 3 724 6

790 6

751 48

751 24 751 3 788 24

714 6

774 48 774 24

618 24

676 24

676 48

751 12 618 6 723 3

-20 -15 -10 -5 0 5 10 15 20

D1 Score

-20

-15

-10

-5

0

5

10

15

20

25

D2

Sco

re

604 24 -2

604 12

604 0 774 0

604 48 676 12

788 0714 3

723 12 788 12

751 0618 0

724 12

604 3

604 24

723 0 676 0

714 12618 12714 24 723 6724 24

724 0 774 3 676 3

714 0

676 6604 12 -2

790 3 723 48

723 24

724 48

618 48

714 48

790 48618 3

774 12

724 3

788 6

774 6

604 6

790 12

790 0

790 24

751 6 788 3 724 6

790 6

751 48

751 24 751 3 788 24

714 6

774 48 774 24

618 24

676 24

676 48

751 12 618 6 723 3

Scores for D1 (27.8 %) versus D2 (24.0 %), Log | Pareto (DA)

036122448

Time point # of features altered

3-48 hours, early sustained

52

48 hours, late transient 28

B)

A)

References:1.Kim MK and JHY Park. (2009) Proceedings of the Nutrition Society; 68:103-10.2.Beaver LM, et al. (2012) Ch.3, In: Diet, nutrition, and cancer.3.Innamorato NG, et al. (2008) Journal of Immunology; 181(1):680-9. 4.Prawan AA, et al. (2008) Pharmaceutical Research; 25(4):837-44.5.Myzak MC, et al. (2007) Experimental Biology and Medicine; 232:227-34.6.Clarke JC, et al. (2011) Pharmacological Research; 64(5):456-63.7.Bitzinger DI, et al. (2008) Cytometry; 73A(7):642-50.8.Harita N, et al. (2009) Diabetes Care; 32(3):424-6.

Acknowledgements:This work was supported by NIH grants CA090890, CA122906, NIEHS grant P30 ES000210. We gratefully acknowledge technical assistance from Lauren Atwell, Dr. Carmen Wong, Dr. Laura Beaver, Karin Hardin and Jeff Morré. Broccoli sprouts were provided by Sprouters NW, Inc.

• Consuming cruciferous vegetables such as broccoli sprouts, kale, Brussels sprouts, and bok choy is associated with several health benefits, including a decreased risk of certain cancers. 1

• Cruciferous vegetables contain the glucosinolate glucoraphanin (GFN).

• Cutting, chopping, or chewing these vegetables releases GFN and the enzyme myrosinase, which transforms GFN into sulforaphane (SFN). 2

• SFN has many demonstrated health benefits in cell and animal models, including anticarcinogenic, antioxidant, and antibacterial properties. 3

• SFN has been shown to target biological pathways leading to:

• apoptosis and the excretion of carcinogens

• cell cycle arrest• eradication of H. pylori infections4,5

• In humans, SFN is metabolized to yield four bioactive metabolites via the mercapturic acid pathway (Fig. 1).

• Specific SFN metabolites may be responsible for some of the health benefits of consuming SFN.

• In humans, SFN metabolites peak in the plasma at 3 hours following consumption and are mostly excreted by 24 hours.6

*

HMDB: Creatinine

Fragment with m/z 114.065

II. Study Goal

III. Significance

IV. Design

VI. Results

VII. Discussion

VIII. Summary and Conclusions

V. Methods V. Methods (continued)

VI. Results (continued)

A)

CH2

SCH3

O

R=

SFNGFN

N C SR

Glu Cys Gly

+

SFN-GSH

NH C SR

S

Glu Cys Gly

SFN-GSH

NH C SR

S

Glu Cys Gly

SFN-Cys

NH C SR

S

Cys

SFN-NAC

NH C SR

S

Cys NACSFN

GST GTP

CGaseNAT

Blood draw

Blood draw

Blood draw

Blood draw

0 hours3 hours6 hours

12 hours24 hours48 hours

Alterations detected in human plasma

metabolome following SFN consumption

Figure 3. Changes in human plasma metabolome were detected following SFN consumption from broccoli sprouts. A) PCA-DA plot showing separation of time points based on all features detected in subjects at each time point. B) Number of features significantly altered (p<0.05, fold change > 2) over time after consuming sprouts (n=10). PCA-DA, principal components analysis – discriminant analysis.

oMS information/settings:• Positive ion mode

o AB SciexTripleTOF™ 5600 mass spectrometeroPeak intensity threshold for MS/MS experiments:

100oCollision energies & mass ranges measured:

•MS only: 10 eV (mass range: 70-1000 m/z)•MS/MS: 40 eV (mass range: 40-1000 m/z)

Metabolite identification:• Student’s t tests were conducted to compare levels of

detected features between time 0 & other time points (3, 6, 12, 24, & 48 hours)

• Fragment spectra for altered features (p<0.05) meeting intensity threshold for MS/MS analysis were compared to MS/MS spectra in metabolomic databases: 1) Metlin: Metabolite and Tandem MS Database, and 2) Human Metabolome Database (HMDB).

• Criteria for candidate metabolite identification:• Endogenous in origin• MS/MS data (i.e., fragment spectra) available in database• Mass tolerances of ≤ 5ppm (Metlin) and 0.01 ppm (HMDB)• Matching m/z ratios of MS/MS fragments between

database & experimentally derived spectra

Creatinine possibly increased 48 hours after SFN consumption

Figure 4. Fragment spectra from MS/MS experiments. Fragment spectra for A) feature with mass-to-charge ratio of 114.065 from study data, and B) creatinine from Human Metabolome Database (HMDB) showing matching m/z values (relative intensity varies from one machine to another).

Biochemical pathways affected may involve creatinine• To our knowledge, SFN has not previously been reported to

alter plasma creatinine levels in humans.• Creatinine plays a large role in muscle metabolism.• High creatinine levels may indicate decreased risk of type 2

diabetes.8

• Molecular pathways involving creatinine may signify a novel mechanism of SFN in promoting human health.

• The plasma metabolome was altered in human subjects following SFN consumption from broccoli sprouts.

• Altered features were observed in the plasma at each time point following sprout consumption.

• Some alterations were transient, while others were sustained through multiple time points.

• Biochemical pathways affected by consumption of broccoli sprouts may involve creatinine.

• Fragment spectra of a feature significantly altered at 48 hours following sprout consumption was visually matched to the fragment spectra of creatinine from the database.

• Future directions: • Additional experiments are needed to validate the feature’s

identity.• More work is needed to understand the full potential of SFN in

human health.