Quantitative Analysis of Carotenoids and DNA Oxidation Products in Human

Tissues Using LC-MS-MS

Richard B. van BreemenDepartment of Medicinal Chemistry and Pharmacognosy

University of Illinois College of PharmacyChicago, Illinois, USA

Lycopene and Prostate Cancer Prevention

Lycopene

• Carotenoid responsible for red color of tomatoes

• Also found in watermelon and pink grapefruit

• Available as a dietary supplement

• Potent antioxidant• Associated with reduced risk

of prostate cancer

-Carotene (pmol) injected on-column

APCI vs Electrospray for LC-MS-MS of Carotenoids

y = 0.0098x - 0.3288R2 = 0.9981

0

5

10

15

20

25

0 500 1000 1500 2000 2500

Mas

s S

pect

rom

eter

Res

pons

e -

Car

oten

e/In

tern

al S

tand

ard APCI

0

0.02

0.04

0.06

0.08

0 500 1000 1500 2000 2500

Electrospray

Negative Ion APCI MS-MS of Lycopene

m /z

467

536

Rel

ativ

e A

bund

ance

[M − C5H 9]-

M –•

100

50

0300 340 380 420 460 500 540 580

Negative Ion Electrospray Product Ion MS-MS with CID of Lycopene and -Carotene

100 200 300 400 500 6000

100

50

467

361269398

536

Rel

ativ

e ab

unda

nce

Lycopene

536

100 200 300 400 500 600m/z

0

100

50

243203189 444430361 458R

elat

ive

abun

danc

e

β-Carotene

M -.

M -.

[M -C 5H 9 ]-.

0 2 4 6 8 100

50

100lycopene

-carotene

0 2 4 6 8 100

50

100

Rel

ativ

e ab

unda

nce

lycopene -carotene

-carotene

0 2 4 6 8 10Retention time (min)

0

50

100lycopene

-carotene

LC-MS SIM of m/z 536

LC-MS, extract spiked with - and -carotene

LC-MS-MS SRM of m/z 536→467 of spiked extract

Fang L, Pajkovic N, Wang Y, Gu C, van Breemen RB. Anal. Chem. 2003, 75: 812-817

LC-MS and LC-MS-MS of the Tomato Dietary Supplement Lyc-O-Mato (LycoRed)

C18 HPLC and Negative Ion APCI

Retention time (min)

0

100

%

0

100

%

Lycopene standardm/z 536 467

Human plasma extractm/z 536 467

MS

-MS

rela

tive

resp

onse all trans-lycopene

5-cis- lycopenecis-lycopenesall trans-lycopene

100

0

%

10.0 20.0 30.0 40.0

Human plasma extract m/z 536

MS

rela

tive

resp

onse

C30 HPLC Negative Ion APCI LC-MS-MS of Lycopene in Human Plasma

Accuracy and Inter-day Precision of LC-MS-MS Analysis of Lycopene (Using the TSQ Quantum)

0.61-0.26498.69 3.0650050

3.90-2.2297.78 3.8110010

5.461.4650.73 2.77505

8.09-7.89.22 0.75101

6.809.365.47 0.3750.5

Inter-day precision(RSD %)

Accuracy(% Deviation)

Measured amount

(mean SD, pmol)

Nominal amount

injected on-column(pmol)

Concentration of standard

solution( M)

* n=5 on five different days

Hypotheses to be Tested

• Lycopene prevents DNA oxidation both in vitro and in vivo.– Does lycopene prevent formation of multiple DNA

oxidation products or only certain products?– Is lycopene a pro-oxidant?– Can in vitro experiments predict in vivo effects of

lycopene?• Oral administration of lycopene results in

increased concentration in the prostate• Serum lycopene reflects prostate levels

Structures of Target Oxidized Deoxynucleosides

NOHO

HO

NHHO

O

O

N

N N

NH

O

NH2

HO

OHO

HO

N

N N

N

NH2

HO

OHO

HO

8-oxo-dG 8-oxo-dA

HMDU

LC-UV-MS-MS Measurement of Oxidized Deoxynucleosides

Precipitate DNAwith 70%

cold ethanol

Isolate DNA from cells or tissue

DNA HydrolysisNuclease P1

Alkaline Phosphatase

Ultrafiltration (no SPE) to remove enzymes

LC-UV-MS-MSAnalysis

UV quantitation of dG, dA, dC & T

MS-MS quantitation of 8-oxo-dG, 8-oxo-dA &

HMDU

LC-UV-MS-MS of Hydrolysate of Fenton-oxidized Calf Thymus DNA

5 10 15 20 250

100

1000

1000

1000

100

Retention time (min)

MS

-MS

rela

tive

resp

onse

A

B

C

D

E

TG 275>116

HMDU9.9 257>141

8-oxo-dG20.1 282>192

8-oxo-dA 267>176

dCT dG dA

UV at 260 nm

26.1

Abso

rban

ce

260

nm

0

Results: Reduction of 8-oxo-dG Levels in Calf Thymus DNA by 1 µg Lycopene

1

2

3

4

5

6

7

0 10 20 30 40 50 60

Incubation time (min)

8-ox

o-dG

leve

l (8-

oxo-

dG/1

05 dG)

without lycopene

with lycopene

Fenton-type reagent: 1 mM FeSO4 /1 mM H2O2

Clinical Study Design

Preliminary Dietary Intervention Study

Tomato sauce (32 men)(equiv. to 30 mg lycopene/d)

Prostate Cancer (randomized, placebo-controlled,

double blind)

Placebo (30 men)

Lycopene 30 mg/day (30 men)

BPH (randomized, placebo-controlled,

double blind)

Placebo (30 men)

Lycopene 30 mg/day (30 men)

• 60 men diagnosed with benign prostate hyperplasia and 60 with prostate cancer who will have prostectomy or biopsy are being recruited (119/120 to date).

• Subjects are randomized into two groups who either take a pill containing lycopene (30 mg) or a placebo every day for 3 wk in a double-blind study.

• Blood samples are obtained at the beginning and end of the intervention period for the measurement of lycopene ,the DNA oxidation products 8-oxo-dG and 8-oxo-dA, and prostate specific antigen (PSA).

• Prostate tissue samples are obtained at the end of the study either from resected tissue or biopsy for the measurement of lycopene and DNA oxidation products.

Human Study Design

• Men with elevated serum PSA were recruited into a tomato sauce whole-foods based study prior to prostate biopsy to confirm prostate cancer. Biopsy and blood samples were obtained for lycopene and DNA oxidation measurement.

• 32 men with stage T1 or T2 adenocarcinoma of the prostate completed the study.

• Subjects consumed 200 g (3/4 cup)/d tomato sauce in pasta dishes for 21-days. (30 mg/d lycopene)

• Total lycopene was measured in serum and prostate tissue obtained at the time of radical prostatectomy.

• 8-oxo-dG was measured in peripheral blood white blood cells and prostate tissue

• PSA levels were measured in serum before and after intervention

Preliminary Clinical Study

Lycopene in Human Serum and Prostate Tissue After Tomato Sauce Intervention

(30 mg lycopene/day)

0

200

400

600

800

1000

1200

1400

Baseline Day 210

0.10.20.30.40.50.60.70.80.9

Baseline Day 21

Ser

um ly

cope

ne (n

M)

Pro

stat

e ly

cope

ne (n

mol

/g)

Prostate Tissue Oxidative DNA Damage in Prostate Cancer Patients After 21-Day

Tomato Sauce Whole Foods Intervention

0

0.2

0.4

0.6

0.8

1

1.2

Referencegroup

Interventiongroup

Prostate

8-ox

o-dG

/105

dG

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Baseline Day 21

Leukocytes

Leukocyte Oxidative DNA Damage (8-oxo-dG)

Serum Prostate Specific Antigen

Serum

PS

A (ng/m

L)Le

ukoc

ycte

8-ox

o-dG

/105

dG

Baseline BaselineAfter intervention

After intervention

0

0.5

1.0

1.5

0

5

10

15

25

20

ConclusionsTomato whole-foods intervention

• Lycopene in serum increased 2-fold• Lycopene levels in prostate tissue increased 3-fold• DNA oxidation (measured as 8-oxo-dG) decreased

21% in leukocytes• DNA oxidation decreased 28% in prostate tissue• PSA in serum decreased 20%• These results are consistent with epidemiological

data showing an inverse correlation between tomato consumption and risk of prostate cancer

Chen, Stacewicz-Sapuntzakis, Duncan, Sharifi, Ghosh, van Breemen, Ashton, Bowen. J. Natl. Cancer. Inst. 2001, 93: 1872-1879.

Conclusions, cont.

• TSQ Quantum provided >50-fold improvement in APCI performance for carotenoid analysis compared to the older model Quattro II it replaced

• Quantum provided 10-fold enhancement of sensitivity for nucleoside analysis using electrospray

• Phase IIb clinical investigations of lycopene (30 mg/d) are in progress in men with prostate cancer or benign prostate hyperplasia (119 out 120 have completed the protocol)

Related Presentations at the 52nd ASMS Conference from this Laboratory

• MPY 469: Liu and van Breemen. Quantitative analysis of monoglutamyl and polyglutamylfolate by hydrophilic interaction LC-MS-MS.

• WPK 182: Zhu and van Breemen. LC-MS-MS determination of TBARs as a measurement of lipid peroxidation in human serum.

• WPP 296: Pajkovic et al. Ultrafiltration LC-MS screening of natural products for ligandsto the androgen receptor.

• And 13 others

Acknowledgements• Wageningen University

– Clive West• University of Illinois at Chicago

– Y Yang, L Yuan, Y Hua, C Gu– N Pajkovic, Y Wang, L Fang– R Morrissy, P Bowen, R Sharifi

• Hunt-Wesson• National Cancer Institute grants

RO1 CA70771 and RO1 CA101052