Reactivity of Chromium(III) Nutritional Supplements in Biological Media: a X-Ray Absorption Spectroscopic Study
Annie Nguyen, Irma Mulyani, Aviva Levina and Peter A. Lay*
School of Chemistry, The University of Sydney, NSW, 2006, Australia
Supporting Information
i
Sample A2 (ANBF)
a
1.5 Raw absorbance Background function
0.008
1.0
0.006
control point
0.004
Normalized absorbance Spline function X-ray crossection (normalized)
0.5
o
0.002
Absorbance
b
0.0
5800
5900
6000
6100
6200
5800
5900
6000
6100
6200
6000
6100
6200
Sample A11 (SSRL) Raw absorbance Background function
0.06
1.5
1.0
Normalized absorbance Spline function X-ray crossection (normalized)
0.04 0.5
control point
0.02
0.0
o 5800
5900
6000
6100
6200
5800
5900
Energy, eV
Figure S1. Typical examples of background subtraction and normalization of XANES spectra, including those with strong (e.g., A2) and weak (e.g., A11) edges, collected at different beamlines (ANBF or SSRL). Designations of the samples correspond to Tables 1-3 (main text). The upper energy limit for data collection was 200 eV above the Cr edge.33 The normalization algorithm, performed in the Spline program within the XFit software package,32 included the following steps: (i) the pre-edge background (red line in a) was fitted with a fourth-order polynomial; (ii) the spline function (red line in b) was set as a first-order polynomial; and (iii) the post-edge part of the background function was adjusted using a control point (a), so that the slope of the resultant spline function corresponded to that of the normalized X-ray crossection for Cr (red and blue lines in b).33 The control points are the points with weight 100 (compared to 1 for the absorbance data points and the k-weighting for the normalised absorbance points) in the least-squares fitting procedure for the corresponding spline curve. They, therefore, act by strongly 'attracting' the spline curve toward themselves and can be used to influence or control the curve.32
ii
1.5
1.5
a
b
A A1 A2
B B1 B2
1.0
1.0 1.4
1.4
0.5
0.5
1.3
1.3
1.2
0.0 5980
Normalized Absorbance
1.5
1.2 6010
6000
6020
6020
0.0 5980 1.5
c
6010
6000
6020
6020
d
A A3 A4
B B3 B4 1.0
1.0 1.4
1.4
0.5
0.5
1.3
1.3
1.2 1.2
6010
5980
1.0
6020
0.0
0.0
1.5
6010
6020
6000
5980
6020
1.5
e A A5 A6 A7 A8
1.0
6000
6020
f B B5 B6 B7
1.4 1.4
0.5
0.5
1.3
1.3
6010
6010
6020
0.0
0.0 5980
1.2
6020
6000
6020
5980
6000
6020
Energy, eV
Figure S2. Comparison of XANES spectra (295 K) of Cr(III) complexes A and B before and after the reactions with artificial digestion systems and blood components. Designations of the samples correspond to Tables 1-3 (main text). The insets are expansions of the main peaks in the XANES.
iii
1.5
a
1.5
b
A A9 A10
B B9 B10
1.0
1.0
1.4
1.4
0.5
0.5 1.3
1.2 1.2
0.0 5980
Normalized Absorbance
1.5
6010
6000
6020
6010
0.0 5980
6020
c
1.5 C C9 C10
6000
6020
6020
d A10 B10 C10
1.0
1.0 1.4
0.5
1.2
1.2 6010
6020
0.0 5980 1.5
1.0
1.4
0.5
1.3
6000
6020
6010
0.0 5980
6000
6020
6020
e A10 L6-Cr(VI) (ANBF) A549-Cr(VI) (SSRL) 1.4
0.5 1.3
6010
6020
0.0 5980
6000
6020
Energy, eV
Figure S3. Comparison of XANES spectra (295 K) of Cr(III) complexes A-C before and after the reactions with cultured mammalian cells or cell culture media. Designations of the samples: L6-Cr(VI) are differentiated rat muscle cells (L6), treated with Cr(VI) (10 µM for 20 h at 310 K); A549-Cr(VI) are human lung carcinoma cells (A549), treated with Cr(VI) (100 µM for 4 h at 310 K; data from Ref. 12); other designations correspond to Tables 1-3 (main text).
iv
1.5
1.0
a
1.5
A11 B11 B11 (smoothed) C11 G
b A-RSA (A11) A-Tr
1.0
1.4 1.4
0.5
0.5 1.3
1.2
1.2
0.0
6020
6010
5980
6000
6020
5980 1.5
c
6010
6000
6020
6020
d
B-RSA (B11) B-Tr
1.5
Normalized Absorbance
0.0
C-RSA (C11) C-Tr 1.0
1.0 1.6
1.4
0.5
1.4
0.5
1.3
1.2 6010
6010
6020
0.0
5980 1.5
1.2
6020
0.0 6000
6020
5980
e
6000
6020
f 1.5
A B C 1.0
D E F G
1.0
1.4
1.6
1.3
0.5
0.5
1.2
6010
1.2
6020
0.0 5980
1.4
6010
0.0 6000
6020
5980
6000
6020
6020
Energy, eV
Figure S4. Comparison of XANES spectra (295 K) of Cr(III)-protein adducts (a-d; RSA is rat serum albumin and Tr is bovine transferrin; see Experimental Section for sample preparation) and of model Cr(III) complexes (e and f) . Designations of the samples correspond to those given inTables 1-3 (main text). v
1
100
a: H2O
75 50 25 0 500
1000
1
100
1500
b: pH ~ 4.0
Relative Abundance (%)
75 50 25 0 500
1000
1
100
1500
c: pH ~ 6.9
75 50 25
2
0 500
2
100 75
4
1000
1500
1
d: pH ~ 8.1
3
50 25 0 500
1000
1500
m/z Figure S5. Typical ESMS signals (positive-ion mode) for a freshly prepared aqueous solution of A (1.0 mM; a) and for the reactions of A (1.0 mM) with NH3/CH3COOH buffer (10 mM) at various pH values for 32 h at 298 K (b-d). Assignments of the main signals (1-4) are given in Table S1.
vi
Table S1. Solutionsa
Assignment of Major ESMS Signals for the Reactions of A with Aqueous Buffer Signal
+m/z
Assignment b
1
663
[Cr3O(O2CEt)6(OH2)3]+
677
[Cr3O(O2CEt)6(OH2)2(MeOH)]+
691
[Cr3O(O2CEt)6(OH2)(MeOH)2]+
704
[Cr3O(O2CEt)6(MeOH)3]+
509
[Cr3O(O2CEt)3(OH)3(OH2)3]+
523
[Cr3O(O2CEt)3(OH)3(OH2)2(MeOH)]+
537
[Cr3O(O2CEt)3(OH)3(OH2)(MeOH)2]+
551
[Cr3O(O2CEt)4(OH)2(OH2)3]+
565
[Cr3O(O2CEt)4(OH)2(OH2)2(MeOH)]+
578
[Cr3O(O2CEt)4(OH)2(OH2)(MeOH)2]+
592
[Cr3O(O2CEt)4(OH)2(MeOH)3]+
3
288
[Cr(O2CEt)3]NH4+
4
196
[Cr2(OH)2(O2CEt)2(OH2)6]2+
2
a
Reactions of A (1.0 mM) with NH3/CH3COOH buffers (10 mM) at pH = 4.0, 6.9 or 8.1 (see Figure S5) for 32 h at 298 K. Designations of the signals (1-4) correspond to Figure S5. b MeOH ligands originate from the flushing solution used for ESMS (H2O:MeOH = 1:1).
vii
0.075
a
Absorbance
0.050
0.000 -0.002
0.025
b
-0.004 5980
6000
0.000
5980
6020
b 5990
6000
6010
6020
6030
Energy, eV
Figure S6. Cr K-edge XANES spectra (raw absorbance data, freeze-dried solids, 295 K) for cell pellets collected after the treatments of L6 cells with C (0.10 mM for 20 h at 310 K). Complex C was added to the cell culture medium either immediately (a) or 24 h prior to (b) the treatment of the cells. Conditions of sample preparation were identical for the both samples. Spectrum (b) was multiplied by a factor of 10, to compensate for the difference in ion chamber gain.
viii
100
a
Monomer fraction Dimer/trimer fraction Polymer fraction
75
50
0
1
, M cm
1
25
300
400
500
600
700
800
100
b Dimer/trimer fraction Before freeze-drying Re-dissolved after freeze-drying
75
50
25
0 300
400
500
600
700
800
, nm
Figure S7. Electronic absorption spectra of Cr(III) aqua-hydroxo complexes:23 (a) chromatographic fractions after the reaction of [Cr(OH2)6]3+ (0.10 M) with NaOH (0.10 M) for 24 h at 298 K, followed by separation on Sephadex G25 gel filtration column (eluted with H2O); and (b) comparison of spectra of the main chromatographic fraction immediately after the separation (black line) and after freeze-drying and re-dissolving in H2O (red line). The extinction coefficients ( , M 1 cm 1) were calculated based on Cr concentrations in solutions, determined by AAS.
ix
Table S2. Characteristic Features in the Electronic Absorption Spectra of Cr(III)-OH-OH2 Complexes Sample
Max a
LW b
Min a
Max a
LW b
Min a
Monomer (deprot.) c
586 (16)
159
511 (8.5)
432 (28)
97
342 (3.7)
Monomer (deprot.) d
590 (16)
NDe
NDe
430 (28)
NDe
NDe
Dimer/trimer c
584 (19)
118
498 (5.3)
422 (24)
79
350 (1.3)
Dimer/trimer c,f
590 (20)
125
498 (4.9)
424 (23)
78
350 (0)
Dimer d
582 (17)
147
490 (5.2)
417 (20)
78
345 (1.5)
Trimer d
584 (19)
121
499 (5.5)
425 (30)
80
346 (2.1)
Tetramer d
580 (16)
115
500 (5.6)
426 (30)
80
347 (1.9)
Hexamer d
585 (19)
129
503 (6.1)
426 (29)
82
353 (2.4)
Polymers c
619 (24)
182
495 (4.8)
428 (14)
87
355 (2.3)
a
Wavelength in nm; extinction coefficients (M 1 cm 1, per Cr ion) are given in parentheses. b Line width at half-height in nm. c Data from this work; samples obtained from the reaction of [Cr(OH2)]6]3+ (0.10 M) with NaOH (0.10 M), followed by separation of the products on a Sephadex G-25 column (see Figure S5 and the Experimental Section in the main text). d Data from Ref. 23. e Data not available. f Sample re-dissolved after freeze-drying.
x
Original Spectra
Experimental Calculated Residual 0.2
A1
Experimental Smoothed Calculated
First Derivatives
A1
1.0 0.1
0.5
0.0
0.0
-0.10 -0.15 5980
6000
6020
5980
0.2
Normalized Absorbance
A2
6000
6020
6000
6020
6000
6020
A2
1.0 0.1 0.5
0.0
0.0
-0.10 -0.15 5980
6000
6020
5980
0.2
A3
A3
1.0 0.1 0.5
0.0
0.0
-0.10 -0.15 5980
6000
6020
5980
Energy, eV
Figure S8. Comparison of experimental and calculated (based on the models listed in Table 3, main text) XANES spectra of biotransformation products of Cr(III) complexes A-C. Designations of the samples correspond to Tables 1-3, main text. Smoothed first-derivative spectra (blue lines) were 35 obtained by the FFT procedure with 20-point window (see the Experimental Section). The figure is continued on the next six pages. The data for the sample A10 are shown in Figure 2, main text.
xi
Original Spectra
Experimental Calculated Residual
Experimental Smoothed Calculated
First Derivatives
A4
A4 0.2 1.0
0.1 0.5
0.0
0.0 -0.10 -0.15 5980
6000
6020
0.2
A5
Normalized Absorbance
-0.1 5980
6000
6020
6000
6020
6000
6020
A5
1.0 0.1 0.5
0.0
0.0
-0.10 -0.15 5980
6000
6020
5980
0.2
A6
A6
1.0 0.1 0.5
0.0
0.0
-0.10 -0.15 5980
6000
6020
5980
Energy, eV
Figure S8 (continued).
xii
Original Spectra
Experimental Calculated Residual
A7
Experimental Smoothed Calculated
First Derivatives
0.2
A7
1.0
0.1 0.5
0.0
0.0
-0.10 -0.15 5980
Normalized Absorbance
1.5
6000
6020
A8
5980
0.2
6000
6020
6000
6020
6000
6020
A8
1.0 0.1 0.5
0.0
0.0
-0.10 -0.15 5980
6000
6020
5980
1.5
A9
A9 0.2 1.0 0.1 0.5 0.0 0.0 -0.1
-0.10 -0.15 5980
6000
6020
5980
Energy, eV
Figure S8 (continued).
xiii
Original Spectra
1.5
Experimental Calculated Residual
B1
Experimental Smoothed Calculated
First Derivatives
0.2
B1
1.0 0.1 0.5 0.0
0.0 -0.10 -0.15 5980
Normalized Absorbance
1.5
6000
6020
-0.1 5980
6000
6020
6000
6020
6000
6020
B2
B2 0.2
1.0 0.1 0.5
0.0
0.0 -0.10 5980
1.5
6000
6020
B3
-0.1 5980
0.2
B3
1.0 0.1 0.5
0.0 -0.1
0.0 -0.10
-0.2
-0.15 5980
6000
6020
5980
Energy, eV
Figure S8 (continued).
xiv
Original Spectra
1.5
Experimental Calculated Residual 0.2
B4
Experimental Smoothed Calculated
First Derivatives
B4
1.0 0.1 0.5 0.0 0.0 -0.10 -0.1 5980
Normalized Absorbance
1.5
6000
6020
5980
6000
6020
6000
6020
6000
6020
B5
B5 0.2
1.0 0.1 0.5 0.0 0.0 -0.10
-0.1
-0.15 5980
1.5
6000
6020
5980
0.3
B6
B6
0.2
1.0
0.1 0.5 0.0 0.0 -0.1
-0.10 -0.15 5980
6000
6020
5980
Energy, eV
Figure S8 (continued).
xv
Experimental Calculated Residual
Original Spectra
1.5
Experimental Smoothed Calculated
First Derivatives
B7
B7 0.2
1.0
0.1 0.5
0.0 0.0 -0.08
-0.1
-0.12 6000
Normalized Absorbance
1.5
6020
B9
5980
0.2
6000
6020
6000
6020
6000
6020
B9
1.0 0.1 0.5
0.0
0.0 -0.10
5980
1.5
6000
6020
B10
5980
0.4
B10
1.0
0.0
0.5
0.0 -0.2 -0.4
-0.4 5980
6000
6020
5980
Energy, eV
Figure S8 (continued).
xvi
Original Spectra
Experimental Calculated Residual
C9
Experimental Smoothed Calculated
First Derivatives
0.2
C9
1.0 0.1 0.5 0.0 0.0 -0.10 -0.1
-0.15 5980
6000
6020
5980
6020
6000
6020
6000
6020
C10
C10
Normalized Absorbance
6000
1.0
0.2
0.5
0.1
0.0
0.0
-0.08 -0.12 5980
6000
6020
5980
L6-Cr(VI)
L6-Cr(VI) 0.2 1.0
0.5
0.1
0.0 -0.09
0.0
-0.12 5980
6000
6020
5980
Energy, eV
Figure S8 (end).
xvii
Exrerimental Calculated
A (solid)
6
0
-6 0
3
6
9
3
6
9
3
6
9
3
6
9
6
9
A1 6
0
-6
0
A2
(k)×k
3
7
0
-7
0
A7 6 0 -6 0
D (solid) 6 0 -6
0
3
k, Å
1
Figure S9. Experimental and fitted XAFS spectra (295 K) of A and D (solid mixtures with BN) and reaction products of A in biological media (A1, A2 and A7, freeze-dried solids). Designations of the samples correspond to Tables 1-3 (main text). Details of the fits are given in Table S3.
xviii
Table S3. Summary of SS XAFS Fitting Resultsa Parameter b k range, Å
A (solid)
A1
A2
1
D
1-11
FT range, Å
0.5-5.0
Ni/p
4.5
R, %
A7
30.0
29.1
31.6
30.7
25.2
E0, eV
3.6(3)
3.6(3)
3.6(3)
3.6(3)
3.6(3)
S0 2 c
0.80(1)
0.81(7)
0.81(7)
0.84(7)
0.81(7)
Shell 1: Cr O/N Nd
6
6
6
6
6
X, Å
1.96(1)
1.96(1)
1.96(1)
1.96(1)
1.98(1)
0.0050(1)
0.0036(4)
0.0020(5)
0.0022(9)
0.0015(2)
2e
, Å2
Shell 2: Cr Cr N
1.9 ± 0.3
2.3 ± 1.0
1.5 ± 0.7
0.7 ± 0.3
--
X, Å
3.24(2)
3.24(2)
3.15(2)
3.10(2)
--
0.0048(6)
0.0043(5)
0.0055(4)
0.0060(6)
--
2e
, Å2
a
Designations of the samples correspond to Tables 1-3 (main text). Errors in the last significant figures (arising from the noise in the data, as calculated by Monte-Carlo analysis) are shown in parentheses. b Designations of the parameters: Ni/p is the determinancy factor (were Ni is the number of independent observations and p is the number of varied parameters);32 R is the goodness-of-fit parameter;32 E0 = E0 – 6005 (eV) is the threshold energy; S02 is the scale factor; N are the numbers of donor atoms in each shell; X (Å) are the average absorber-scatterer distances; and 2 (Å2) are the DebyeWaller factors. c Restrained to be within the 0.80-1.0 range.32 d The values were not varied during the optimization. e Restrained to be within the 0.0005-0.02 Å2 range.
xix
Reaction in serum Reaction in HBS
A (l = 1 cm)
0.15
0.10
Cr(VI)
0.05
0.00 400
600
800
, nm
Figure S10. Typical electronic absorption spectra of the reaction mixtures containing B (1.0 mM) and H2O2 (5.0 mM) in undiluted rat serum or HEPES-buffered saline (HBS, pH = 7.4) after 1 h of reaction at 310 K. The reaction mixtures were diluted 10-fold with H2O prior to the spectral measurement.
xx
