Supplemental Information
Article title: Quantification of 1 H NMR Spectra from Human Plasma
Journal: Metabolomics
Authors:
Robin A. de Graaf 1 , Hetty Prinsen 1 , Cosimo Giannini 2 , Sonia Caprio 3 , Raimund I. Herzog 4
Affiliations:
Departments of Diagnostic Radiology 1 , Pediatrics 3 and Internal Medicine 4
Magnetic Resonance Research Center
Yale University School of Medicine
New Haven, Connecticut, USA
Department of Pediatrics 2
University of Chieti
Chieti, Italy
Corresponding author email: robin.degraaf@yale.edu
1

Table S1: Metabolite concentrations (in  M) obtained with diffusion NMR or ultrafiltration metabolite sample
concentration (uM) diffusion ultrafiltration delta (%)
2-hydroxy butyrate
3-hydroxy butyrate acetate acetoacetate acetone alanine arginine betaine
5
1
3
4
2
5
1
3
4
2
3
4
5
5
1
3
4
2
5
1
3
4
2
5
1
2
3
4
1
2
3
4
1
2
5
3
4
1
2
5
256.31
265.25
250.65
23.90
75.65
54.61
29.01
32.89
52.92
44.39
3.46
4.37
4.67
63.16
39.18
32.26
24.64
34.53
278.57
240.09
58.75
60.56
59.02
60.39
69.53
69.35
66.08
76.75
2.35
7.93
36.92
23.55
26.27
25.33
19.36
83.01
44.84
44.24
54.11
47.82
247.92
256.74
237.02
43.50
45.76
56.94
56.61
32.45
64.09
54.51
4.48
4.01
4.61
46.56
48.05
33.63
35.77
26.23
265.67
230.11
68.88
61.26
61.40
81.89
79.12
78.22
65.11
93.86
3.12
7.29
43.82
42.49
32.15
27.50
24.56
119.58
40.65
30.99
40.21
39.08
-3.33
-3.26
-5.59
58.17
-49.24
4.18
64.46
-1.34
19.09
20.46
25.81
-8.72
-1.31
-30.27
20.33
4.16
36.87
-27.32
-4.74
-4.24
15.87
1.16
3.95
30.22
12.90
12.01
-1.48
20.05
28.11
-8.35
17.11
57.35
20.13
8.23
23.67
36.10
-9.81
-35.23
-29.48
-20.13
Table S1 continued
2

creatinine ethanol glucose glutamate glutamine carnitine choline citrate creatine
2
3
5
1
4
2
3
5
1
4
2
3
5
1
4
2
3
4
4
5
2
3
1
4
5
2
3
1
4
5
1
1
2
3
5
1
2
2
3
5
1
4
3
4
5
31.96
102.00
71.34
104.15
67.01
62.29
4.92
7.55
6.11
6.22
52.69
53.02
52.12
58.33
41.18
35.11
32.72
35.92
29.23
30.80
40.55
34.77
38.49
104.99
76.73
87.56
76.26
112.35
168.96
146.21
175.72
167.44
119.36
49.41
6.00
94.17
90.33
93.13
76.05
111.58
467.26
408.72
398.37
387.14
342.31
1.53
140.29
86.68
37.57
125.81
117.61
0.00
0.00
0.00
0.00
-5.12
4.63
-3.30
2.36
1.64
-7.30
1.55
-0.89
21.86
15.32
7.86
22.58
17.41
10.39
-1.90
1.69
3.40
-31.48
3.99
17.71
-8.56
-3.52
1.62
-5.10
0.00
-11.37
-18.41
-11.64
5.29
-28.47
-8.66
4.46
-12.86
-6.28
-9.29
Table S1 continued
32.45
581.27
180.48
152.33
294.28
240.12
4.92
7.55
6.11
6.22
50.05
55.53
50.43
59.72
41.87
32.63
33.23
35.60
36.41
35.91
43.87
43.62
45.83
116.49
75.29
89.05
78.90
81.80
175.84
174.62
161.29
161.64
121.31
46.95
6.00
84.04
75.11
82.89
80.19
83.77
428.49
427.38
350.22
363.58
311.91
3

methionine ornithine proline pyruvate succinate isoleucine lactate leucine lysine
2
3
5
1
4
2
3
5
1
4
2
3
5
1
4
2
3
4
4
5
2
3
1
4
5
2
3
1
4
5
1
1
2
3
5
1
2
2
3
5
1
4
3
4
5
15.16
56.12
29.22
9.22
33.16
18.66
132.73
199.08
138.94
109.37
77.91
80.87
98.78
86.32
20.27
14.95
17.97
13.82
58.73
39.74
34.35
31.11
28.51
1053.19
1280.48
1471.78
1407.16
1260.83
131.17
106.54
105.96
93.34
90.79
119.93
120.38
5.98
25.59
26.96
30.62
32.43
9.39
9.74
7.60
7.14
5.37
-10.90
25.39
-7.66
27.03
26.53
-28.01
13.08
-16.75
-27.56
-1.78
26.56
14.99
14.19
19.36
32.86
17.78
-35.86
-11.21
-2.34
1.10
-1.12
1.62
-0.60
-4.65
-3.06
13.92
1.49
22.54
17.50
22.52
11.34
18.71
5.42
4.78
-21.81
19.08
-18.17
14.51
-6.47
3.41
-2.66
-56.15
-87.19
-38.95
-68.38
Table S1 continued
13.60
72.44
27.06
12.10
43.30
14.08
151.30
168.31
105.29
107.45
101.78
93.98
113.86
104.82
28.24
17.87
12.51
12.35
69.99
49.83
38.48
37.53
30.09
1104.74
1250.90
1487.98
1391.46
1281.47
130.38
101.69
102.76
107.30
92.15
150.40
96.71
7.24
21.33
31.18
28.71
33.55
9.14
5.47
2.99
4.81
2.63
4

threonine valine
4
5
1
1
2
3
4
5
2
3
230.46
110.30
253.91
245.46
209.49
233.77
191.42
175.29
162.30
153.06
251.82
88.36
272.83
267.40
228.06
225.34
186.63
177.88
165.79
154.44
8.86
-22.08
7.18
8.56
8.49
-3.67
-2.53
1.46
2.13
0.89
Footnotes:
1.
Glucose is used as an internal concentration reference and is therefore by definition identical between diffusion NMR and ultrafiltration.
2.
Delta (%) is defined as 100 x ([m] filtration
– [m] diffusion
)/ [([m] filtration
+ [m] diffusion
)/2] whereby
[m] represent the concentration of metabolite m.
5

90
370
45
1000
100
90
15
30
50
35
220
150
50
30
6000
6000
140
25
10
200
180
35
55
50
10
50
240
45
2HB
3HB
Acetate
Acetoacetate
Acetone
Alanine
Arginine
Betaine
Carnitine
Choline
Citrate
Creatine
Creatinine
GlucoseA
GlucoseB
Glutamate
Glutamine
Isoleucine
Lactate
Leucine
Lysine
Methionine
Ornithine
Proline
Pyruvate
Succinate
Threonine
Valine
Footnotes:
Table S2: Monte-Carlo simulation results metabolite conc (uM) conc (uM) SD (%) no lipoprotein baseline
34.59
54.62
3.72
1.63
49.78
10.16
49.93
240.35
43.78
1.11
5.30
1.13
0.42
3.66
89.63
369.86
44.97
999.65
99.91
89.55
14.70
30.93
50.27
34.68
217.93
149.45
49.97
29.76
6000.00
6000.00
140.11
25.11
9.96
200.46
179.84
0.99
3.14
5.68
0.88
0.24
2.01
0.55
1.64
0.16
0.44
2.06
2.53
3.46
0.35
0.32
0.80
0.54
0.93
1.29
0.00
0.00
conc (uM) lipoprotein baseline
SD (%)
34.76
55.90
3.87
1.76
49.89
9.82
50.12
239.61
43.28
1.05
2.72
2.34
0.50
4.66
50.29
34.84
218.80
149.48
50.02
30.41
6000.00
6000.00
89.63
368.11
45.18
1000.26
99.99
89.33
14.58
29.03
140.17
25.19
9.86
199.74
179.65
1.58
2.50
5.74
0.75
0.60
1.63
0.54
2.39
0.16
0.58
1.06
3.93
4.81
0.53
0.52
0.77
0.50
0.69
1.49
0.00
0.00
conc (uM)
100 uM ethanol
33.67
101.97
49.79
8.61
49.82
239.29
43.30
82.16
365.95
48.26
997.10
99.46
88.21
14.74
27.05
50.08
34.35
216.31
148.63
49.82
29.92
6000.00
6000.00
129.93
23.54
8.18
201.85
179.29
SD (%)
1.66
0.52
1.62
0.13
0.47
1.15
3.07
4.37
0.51
0.34
0.84
0.38
0.54
1.47
0.00
0.00
1.16
2.70
6.20
0.63
0.45
3.19
1.82
0.87
2.87
1.29
0.48
3.92
1.
All concentrations represent the average and SD of 10 Monte-Carlo simulations.
2.
In the presence of an ‘unknown’ 100 uM ethanol signal, only the concentration of the directly overlapping signal from 3HB is significantly changed (red, column 7).
3.
Typical results and details associated with columns 3/4, 5/6 and 7/8 can be found in
Figures S1, S2 and S3, respectively.
6

Figure S1: Spectral fitting result of a single Monte-Carlo simulation. The 1 H NMR spectrum (blue line) was created using the simulated basis set signals scaled by the concentrations in column 2 of Table S2. The lipoprotein baseline was not included in this simulation. The spectral noise level was set equal to that obtained in the experimental 1 H NMR spectra. The spectral fit (red line) is accurate to within the spectral noise level, which is also confirmed by the concentrations listed in Table S2 (columns 3 and 4).
7

Figure S2: Spectral fitting result of a single Monte-Carlo simulation. The 1 H NMR spectrum (blue line) was created using the simulated basis set signals scaled by the concentrations in column 2 of Table S2. The lipoprotein baseline was added by using the parameters obtained on sample 2.
The spectral noise level was set equal to that obtained in the experimental 1 H NMR spectra. The spectral fit (red line) is accurate to within the spectral noise level, which is also confirmed by the concentrations listed in Table S2 (columns 5 and 6). This underlines that the addition of a lipoprotein baseline does not fundamentally change the quantitative nature of NMR.
8

Figure S3: Spectral fitting result of a single Monte-Carlo simulation. The 1 H NMR spectrum was created as described for Figure S1 with the exception that 100

M ethanol was added to the spectrum, but not to the spectral basis set. The fitting result (red line) is largely identical to that shown in Fig. S1, except that the ethanol signal appear in the difference spectrum. As 3HB is overlapping with ethanol, its concentration has been significantly altered (columns 7 and 8).
However, the concentration of most other signals remain unperturbed to with the standard deviation. Visual inspection of the spectral difference (green line) immediately reveals that the spectral basis set was incomplete. Inclusion of ethanol into the spectral basis set provides a highquality fit similar to that shown in Figure S1 and restoring the fitted concentrations.
9

10

11