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19

Figure 1 Formation of the oxidized metabolites of thiamine.

A, Total LC-ESI-MS/MS ion chromatograms of reaction mixtures of thiamine with ROS/ROS

donors. Thiamine (1 mM) was incubated with HOCl (100 μM), H2O2 (100 μM), EP reagent (100

μM), AAPH (100 μM), or Fe2+ (5 μM) /ascorbic acid (AsA) (100 μM) for 24 h at 37 oC. B,

Chemical structures of thiamine and its oxidized metabolites.

Figure 2 Co-injection experiments on the LC-ESI-MS/MS of authentic oxidized thiamine

metabolites with the reaction mixture of thiamine with HOCl.

A, Co-injection of authentic FAP and the reaction mixture of thiamine with HOCl. SRM for FAP

10

(m/z 167 > 122). B, Co-injection of authentic TSA and the reaction mixture of thiamine with

11

HOCl. SRM for TSA (m/z 331 > 122). C, Co-injection of authentic TSE and the reaction mixture

12

of thiamine with HOCl. SRM for TSE (m/z 297 > 122).

13

14

Figure 3 HOCl scavenging activity of thiamine.

15

HOCl scavenging activity was evaluated by measuring the residual HOCl in the reaction mixture.

16

HOCl (100 μM) was incubated with 100 μM of thiamine, TPP, cysteine, or methionine for 5min.

17

18

Figure 4 Quantification of the oxidized thiamine metabolites by LC-ESI-MS/MS with SRM

19

mode.

20

A, Proposed fragmentation pattern of FAP (upper) and FAP-d3 (lower). B, LC-ESI-MS/MS

21

analysis of FAP. Upper, SRM for FAP (m/z 167 > 122); lower, SRM for FAP-d3 (m/z 170 > 125).

22

C, Proposed fragmentation pattern of TSA (upper) and [13C3]-TSA (lower). Asterisk indicates 13C

23

label. D, LC-ESI-MS/MS analysis of TSA. Upper, SRM for TSA (m/z 331 > 122); lower, SRM

24

for [13C3]-TSA (m/z 334 > 122). E, Proposed fragmentation pattern of TSE (upper) and [13C3]-TSE

25

(lower). Asterisk indicates 13C label. F, LC-ESI-MS/MS analysis of TSE. Upper, SRM for TSE

26

(m/z 297 > 122); lower, SRM for [13C3]-TSE (m/z 300 > 122).

27

28

Figure 5 Quantitative analysis of the oxidized thiamine metabolites generated by the

29

reaction of thiamine with HOCl.

30

A, HOCl-concentration-dependent formation of the oxidized thiamine products. Thiamine (1 mM)

31

was incubated with the indicated concentrations of HOCl (0-250 μM) for 24 h at 37 oC. B, Time-

32

dependent formation of the oxidized thiamine products. Thiamine (1 mM) was incubated with

33

HOCl (100 μM) for the indicated times (0-24 h) at 37 oC. Thiamine and its oxidized products

34

were quantified by LC-ESI-MS/MS with SRM mode. Data are mean ± SD of three independent

35

experiments.

20

Figure 6 Formation of the oxidized thiamine metabolites in the activated neutrophil-like

cells.

A, Time-dependent formation of the oxidized thiamine metabolites. The neutrophil-differentiated

HL-60 cells were stimulated with PMA (100 nM) for indicated times (0-6 h), and the resulting

culture supernatants were analyzed by LC-ESI-MS/MS with SRM mode. B, Effect of MPO

inhibitor 4-ABH on PMA-induced formation of oxidized thiamine metabolites in the neutrophil-

differentiated HL-60 cells. The cells were stimulated with PMA (100 nM) together with or

without 4-ABH (100 μM) for 6 h. The culture supernatants were analyzed by LC-ESI-MS/MS in

10

the SRM mode. ***, p < 0.005. N.D., not detected. Data are mean ± SD of three independent

11

experiments.

12

13

14

Figure 7 Formation of the oxidized thiamine metabolites in the LPS-stimulated mouse lung

15

tissue.

16

A, MPO expression ratio in the lung tissues of the LPS-treated mice. The expression levels of

17

MPO and β-actin were analyzed by western blot. Values are mean ± SD (n = 6). ***, p < 0.005.

18

B, Quantification of thiamine and its oxidized metabolites in the lung tissues of the LPS-

19

stimulated or vehicle control mice. Values are mean ± SD (n = 6). **, p < 0.01

20

21

Figure 8 A proposed mechanism for the formation of oxidized thiamine metabolites derived

22

from the reaction of thiamine and HOCl.

23

Thiol derived from thiamine could be converted to FAP via thioketone. Sulfenyl chloride could be

24

converted to TSST or TSE. Furthermore, TSST reacts with HOCl and could be transformed to

25

TSA or TSE.

26

21

Thiamine

+Fe2+/AsA

Thiamine

+ AAPH

NH2

Intensity (×108)

+ EP

NH

FAP (peak A)

+H2O2

TSST

+ HOCl

TSA (peak B)

TSST

NH2

Control

Retention time (min)

S O

TSE (peak C)

1.5

Authentic FAP

0.5

Authentic TSA

Thiamine/HOCl

reaction mixture

10

25

Authentic TSA

20

15

10

10

Thiamine/HOCl

reaction mixture

Relative intensity (x106)

Relative intensity (x107)

Relative intensity (x107)

12

0.2

Thiamine/HOCl

reaction mixture

0.1

Retention time (min)

Authentic TSE

Thiamine/HOCl

reaction mixture

Relative intensity (x107)

14

10

Relative intensity (x107)

Authentic FAP

Thiamine/HOCl

reaction mixture

Relative intensity (x106)

Relative intensity (x106)

Relative intensity (x107)

Relative intensity (x107)

Authentic TSE

0.8

Thiamine/HOCl

reaction mixture

0.6

0.4

0.2

Retention time (min)

Retention time (min)

100

HOCl (μM)

80

60

40

20

NH2

NH

FAP (m/z 167)

NH

NH2

m/z 122

m/z 122

[13C3]-TSA (m/z 334)

FAP-d3 (m/z 170)

FAP-d3

m/z 170>125

Retention time (min)

TSA

m/z 331>122

Relative intensity (x105)

Relative intensity (x105)

Relative intensity (x106)

S O

[13C

3]-TSA

m/z 334>122

0.5

Retention time (min)

TSE

m/z 297>122

Relative intensity (x105)

FAP

m/z 167>122

Relative intensity (x105)

[13C3]-TSE (m/z 300)

Relative intensity (x105)

m/z 125

S O

TSE (m/z 297)

TSA (m/z 331)

NH2

m/z 122

m/z 122

m/z 122

D3C

NH2

1.2

[13C3]-TSE

m/z 300>122

0.8

0.6

0.4

0.2

Retention time (min)

1000

80

800

600

200

40

50 100 150 200 250

HOCl (µM)

40

1000

900

800

50

50 100 150 200 250

HOCl (µM)

30

20

10

12 18

Time (h)

24

1.5

0.6

0.5

0.0

50 100 150 200 250

HOCl (µM)

0.4

0.2

0.0

50 100 150 200 250

15

TSST (μM)

40

20

50 100 150 200 250

HOCl (µM)

12

18

24

10

10

Time (h)

20

12

18

24

HOCl (µM)

50

30

TSE (μM)

0.8

TSA (μM)

1.0

Time (h)

2.0

TSE (μM)

TSA (μM)

60

20

TSST (μM)

FAP (μM)

100

Thiamine (μM)

1200

FAP (μM)

Thiamine (μM)

12 18

Time (h)

24

12 18

Time (h)

24

110

Thiamine (μM)

FAP (μM)

Thiamine (μM)

Time (h)

100

90

0.5

0.0

50

TSE (nM)

TSE (nM)

50

Control PMA 4-ABH

PMA

***

TSA (nM)

***

100

***

1.0

Control PMA 4-ABH

PMA

150

TSA (nM)

***

80

10

Time (h)

1.5

FAP (μM)

40

***

***

30

20

10

N.D.

Control PMA 4-ABH

PMA

Time (h)

Time (h)

200

***

TSST (nM)

TSST (nM)

***

150

100

50

Time (h)

Control PMA 4-ABH

PMA

Control PMA 4-ABH

PMA

MPO relative expression ratio

(MPO/β-actin)

***

0.8

0.4

Control LPS

FAP

(pmol/g tissue weight)

Thiamine

(pmol/g tissue weight)

1.2

0.0

Control LPS

40

**

30

20

10

Control LPS

TSE

(pmol/g tissue weight)

50

**

Control LPS

Cl

HOCl H2O

+H2O

-H2O

Thiamine

Thiol

Thioketone

HOCl

hydrolysis

H2 O

HCl

+ Thiamine

TSST

Sulfenyl chloride

FAP

HOCl

HCl

HCl

TSA

TSE

Sulfinyl chloride

Supplementary Data

Quantitative analysis of oxidized vitamin B1 metabolites generated

by hypochlorous acid

Hitoshi Sasatsuki1, Atsuo Nakazaki1, Koji Uchida2,3, and Takahiro Shibata1,4 *

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601,

Japan

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo

113-8657, Japan

Japan Agency for Medical Research and Development, CREST, Tokyo, Japan

Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya

University, Nagoya 464-8601, Japan

To whom correspondence should be addressed. Takahiro Shibata, Ph.D., Graduate

School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Tel:

81-52-789-4125, Fax: 81-52-789-5741 E-mail: shibatat@agr.nagoya-u.ac.jp

Running title: Quantitative analysis of oxidized vitamin B1 metabolites

Table S1

Analyte

Thiamine

FAP

TSE

TSST

TSA

Validation data in cell culture medium

Validation level

Accuracy

Precision

(nM)

(Bias %)

(RSD %)

156.3

-3.8

2.2

1250.0

-1.1

1.7

10000.0

5.1

3.1

2.0

-7.3

3.5

15.6

1.6

4.9

125.0

2.1

4.2

0.4

9.2

11.8

3.1

1.8

9.1

25.0

-0.9

6.2

6.0

-5.3

6.1

15.6

-6.1

4.0

125.0

-0.7

3.1

11.9

6.8

10.4

31.3

8.3

7.7

250.0

5.0

5.7

Table S2 Relative matrix effects experimental results of three different lots of cell

culture medium

Analyte

Thiamine

Matrix effect (%)

100.8

FAP

99.2

TSE

97.1

TSST

99.4

TSA

97.7

Table S3

Analyte

Thiamine

FAP

TSE

Validation data in mouse lung tissue homogenate

Validation level

Accuracy

Precision

(nM)

(Bias %)

(RSD %)

15.6

3.4

3.0

125.0

3.2

3.0

1000.0

2.4

2.8

1.6

8.6

8.7

12.5

1.8

3.1

100.0

0.7

3.3

0.3

-0.6

5.5

0.8

17.4

7.6

6.0

-4.0

5.0

Table S4

Relative matrix effects experimental results of three different lots of

mouse lung tissue homogenate

Analyte

Matrix effect (%)

Thiamine

105.0

FAP

100.4

TSE

83.3

Figure S1

position

δH (ppm)

δC (ppm)

2.50 (s, 3H)

21.4

165.0

163.0

111.7

7.99 (s, 1H)

144.4

4.31 (s, 2H)

35.0

8.20 (s, 1H)

170.9

Figure S1 The 1H- and 13C-NMR spectroscopic data of peak A in D2O.

Figure S2

position

δH (ppm)

δC (ppm)

2.51 (s, 3H)

20.9

164.2

162.2

111.0

7.96 (s, 1H)

142.8

4.20-4.70 (m, 2H)

39.5

8.08 (s, 1H)

165.6

136.5

138.5

10

2.06 (s, 3H)

18.5

11

2.70 (t, J = 7.4Hz, 2H)

33.1

12

3.73 (t, J = 7.4Hz, 2H)

60.3

Figure S2 The 1H- and 13C-NMR spectroscopic data of peak B in D2O.

Figure S3

position

δH (ppm)

δC (ppm)

2.52 (s, 3H)

17.8

159.6

160.8

106.4

8.05 (s, 1H)

140.6

4.59 (d, J = 16.0 Hz, 1H)

36.0

6’

4.82 (d, J = 16.0 Hz, 1H)

36.0

8.34 (s, 1H)

162.6

133.7

149.8

10

2.14 (s, 3H)

14.5

11

2.81-3.06 (m, 2H)

23.2

12

4.81-4.86 (m, 2H)

72.3

Figure S3 The 1H- and 13C-NMR spectroscopic data of peak C in D2O.

Figure S4

TSA

TSE

Figure S4 The HMBC correlations in the structure of TSA and TSE.

Figure S5

TPP

TPP

+ HOCl

FAP

TPP

Intensity (×107)

TPP

Thiamine

Thiamine

+ HOCl

TSE

TSA

TSST

FAP

Thiamine

Thiamine

Retention time (min)

Figure S5 Total LC-ESI-MS/MS ion chromatograms of reaction mixtures of

thiamine/TPP with HOCl

Thiamine or TPP (1 mM) was incubated with HOCl (100 μM) for 24 h at 37 oC.

Figure S6

40

y = 0.0284x - 0.1436

R² = 0.9990

30

20

10

250

500

750 1000

FAP (nM)

Peak area

(analyte/Internal standard)

Peak area

(analyte/Internal standard)

25

y = 0.022x + 0.3112

R² = 0.9974

20

15

10

250

500

750 1000

TSA (nM)

Peak area

(analyte/Internal standard)

60

y = 0.1242x + 1.0399

R² = 0.9958

40

20

100

200

300

400

TSE (nM)

Figure S6 Calibration curves for the oxidized thiamine metabolites

A, Calibration curve for FAP. B, Calibration curve for TSA. C, Calibration curve for

TSE

Figure S7

Relative intensity (x105)

m/z 122

Thiamine (m/z 265)

Thiamine

m/z 265>122

Relative intensity (x105)

m/z 125

Thiamine-d3 (m/z 268)

Thiamine-d3

m/z 268>125

Retention time (min)

Peak area

(analyte/Internal standard)

200

y = 8.451x - 0.1252

R² = 0.9998

150

100

50

10

15

20

Thiamine (μM)

Figure S7 Quantification of thiamine by LC-ESI-MS/MS with SRM mode.

A, Proposed fragmentation pattern of thiamine (upper) and thiamine-d3 (lower). B, LCESI-MS/MS analysis of thiamine. Upper, SRM for thiamine (m/z 265 > 122); lower,

SRM for thiamine-d3 (m/z 268 > 125). C, calibration curve for thiamine.

Figure S8

m/z 124

TSST (m/z 282)

Relative intensity (x105)

TSST

m/z 282>124

Relative intensity (x105)

m/z 127

TSST-d6 (m/z 285)

TSST-d6

m/z 285>127

Retention time (min)

Peak area

(analyte/Internal standard)

y = 0.0137x - 0.0058

R² = 1.0000

50 100 150 200 250

TSST (nM)

Figure S8 Quantification of TSST by LC-ESI-MS/MS with SRM mode.

A, Proposed fragmentation pattern of TSST (upper) and TSST-d3 (lower). B, LC- ESIMS/MS analysis of thiamine. Upper, SRM for TSST (m/z 282 > 124); lower, SRM for

TSST-d6 (m/z 285 > 127). C, calibration curve for TSST.

Figure S9

***

40

***

PMA

MPO

30

6 (h)

80 kDa

60 KDa

50 kDa

HOCl (μM)

Time

20

10

Control PMA

4-ABH

PMA

Figure S9 Activation of neutrophil-like HL60 cells by PMA

A, Immunoblot analysis of MPO in culture media of HL60 cells. The neutrophildifferentiated HL-60 cells were stimulated with PMA (100 nM) for indicated times (0-6

h), and the resulting culture supernatants were analyzed by immunoblot analysis with

anti-MPO antibody. B, Quantification of HOCl in culture media of HL60 cells. After

treatment of the neutrophil-differentiated HL-60 cells with PMA (100 nM) together

with or without 4-ABH (100 μM) for 6 h, the concentration of HOCl in culture media

was analyzed as described in “Experimental Procedures”.

...