Supplemental material to:Discrimination of steatosis and NASH in mice using nuclear magnetic resonance spectroscopy

Matthias S. Klein1, Christoph Dorn2, Michael Saugspier2, Claus Hellerbrand2, Peter J. Oefner1, Wolfram Gronwald1*

1Institute of Functional Genomics, University of Regensburg, Josef-Engert-Strasse 9, 93053 Regensburg, Germany and 2Department of Internal Medicine I, University Clinic of Regensburg, 93042 Regensburg, Germany.

Supplemental Figure S1.Principal component analysis of urinary NMR spectra. BALB/c mice each fed either a standard chow (circles), or diets inducing simple steatosis (squares) and NASH (triangles), respectively. The filled and empty circles represent the first and second cohort of the control group, respectively.

Supplemental Figure S2.ICA reconstructions based only on the 25 best bins of the 2 used independent components. (A) lipophilic liver extracts, (B) hydrophilic liver extracts, (C) urine. Circles = control, squares = simple steatosis, triangles = NASH.
Supplemental Table S1: Solvent areas excluded from analysis

Sample / Type / 1H-Area [ppm] / 13C-Area [ppm]
Urine / 1D / 6.2-4.7 / -
Urine / 2D / 6.2-4.0 / 150-5
Aqueous extracts / 1D / 4.9-4.7 / -
Aqueous extracts / 2D / 5.6-4.0 / 150-5
Lipophilic extracts / 1D / 7.6-6.9 / -
Lipophilic extracts / 2D / 7.6-6.9 / 85-75

Supplemental Table S2: Frequencies used for spectra of lipophilic liver extracts. *Signals used for the estimation of the total lipid content.

Name / Frequency [ppm] / Functional Group / Molecular mass
Lipid Methene* / 5.45 - 5.20 / -CH=CH- / 26.0 (2H + 2C)
Lipid Diallylic* / 2.90 - 2.74 / =CH-CH2-CH= / 14.0 (2H + C)
Lipid Allylic* / 2.14 - 1.93 / -CH2-CH=CH- / 14.0 (2H + C)
Lipid Methylene* / 1.41 - 1.20 / -CH2- / 14.0 (2H + C)
Lipid Methyl* / 0.94 - 0.80 / -CH3 / 15.0 (3H + C)
Lipid α-Methylene to carboxyl* / 4.325 - 4.260 / COO-CH2-CH2- / 58.0 (2H + 2C + 2O)
Lipid Glycerine* / 4.325 - 4.260 / 41.0 (5H + 3C)
4.175 - 4.110
Cholesterol / 1.01 - 1.008 / not used
Cholesterol ester / 1.021 - 1.017 / not used

Supplemental Table S3: Loadings of IC1 of lipophilic liver extracts

Factor / Frequency / Compound / P-value
Control / NAFLD / P-value
Control / NASH / P-value
NAFLD / NASH
-1.51E-02 / 1.255 / (CH2)n / 4.25E-05 / 2.73E-04 / 1.80E-07
-4.12E-03 / 1.265 / (CH2)n / 8.86E-05 / 2.51E-07 / 9.28E-02
-2.66E-03 / 1.275 / (CH2)n / 4.86E-04 / 9.46E-08 / 7.68E-01
2.38E-03 / 0.675 / Cholesterol (total) / 1.18E-02 / 4.18E-05 / 4.15E-05
-2.20E-03 / 1.245 / (CH2)n / 9.76E-03 / 1.11E-01 / 3.69E-04
-2.06E-03 / 1.285 / (CH2)n / 5.60E-03 / 2.21E-03 / 7.62E-02
2.03E-03 / 1.015 / Cholesterol ester / 2.21E-02 / 7.65E-05 / 6.57E-05
1.70E-03 / 0.865 / CH3 / 1.34E-04 / 6.60E-06 / 5.53E-06
1.54E-03 / 0.855 / CH3 / 4.11E-01 / 4.16E-05 / 1.21E-05
1.25E-03 / 0.905 / CH3 / 9.26E-11 / 5.14E-02 / 4.53E-05
1.18E-03 / 0.915 / CH3 / 7.20E-05 / 5.59E-05 / 3.06E-05
-1.13E-03 / 0.885 / CH3 / 3.95E-02 / 5.10E-01 / 2.46E-02
9.18E-04 / 1.025 / Cholesterol ester / 1.86E-01 / 1.96E-05 / 1.23E-05
8.90E-04 / 1.345 / (CH2)n / 1.09E-06 / 3.34E-06 / 3.18E-02
8.24E-04 / 1.305 / (CH2)n / 1.85E-02 / 4.29E-04 / 7.95E-01
8.07E-04 / 2.265 / (CH2COO) / 8.04E-02 / 5.53E-06 / 1.54E-05
7.85E-04 / 1.355 / (CH2)n / 8.00E-09 / 9.61E-08 / 3.13E-01
7.48E-04 / 1.135 / ? / 2.88E-01 / 3.19E-05 / 2.18E-05
-7.46E-04 / 2.005 / CH2C=C / 1.84E-06 / 9.87E-09 / 5.04E-02
6.90E-04 / 0.925 / CH3 / 1.52E-02 / 1.83E-05 / 5.45E-06
6.82E-04 / 1.335 / (CH2)n / 7.66E-02 / 1.31E-02 / 1.65E-02
-6.72E-04 / 2.015 / CH2C=C / 7.28E-08 / 2.87E-09 / 1.02E-02
-6.51E-04 / 1.295 / (CH2)n / 3.69E-01 / 3.46E-02 / 8.55E-01
6.23E-04 / 1.845 / (CH2CH=CH) / 1.24E-01 / 3.14E-05 / 2.48E-06
6.10E-04 / 0.875 / CH3 / 3.58E-01 / 3.83E-05 / 2.21E-03

Supplemental Table S4: Loadings of IC2 of lipophilic liver extracts

Factor / Frequency / Compound / P-value
Control / NAFLD / P-value
Control / NASH / P-value
NAFLD / NASH
-6.19E-03 / 1.265 / (CH2)n / 8.86E-05 / 2.51E-07 / 9.28E-02
-5.81E-03 / 1.275 / (CH2)n / 4.86E-04 / 9.46E-08 / 7.68E-01
4.74E-03 / 1.255 / (CH2)n / 4.25E-05 / 2.73E-04 / 1.80E-07
-3.70E-03 / 0.865 / CH3 / 1.34E-04 / 6.60E-06 / 5.53E-06
3.58E-03 / 2.055 / CH2C=C / 7.16E-03 / 6.93E-10 / 1.02E-01
-3.55E-03 / 2.015 / CH2C=C / 7.28E-08 / 2.87E-09 / 1.02E-02
-3.55E-03 / 2.005 / CH2C=C / 1.84E-06 / 9.87E-09 / 5.04E-02
3.44E-03 / 2.045 / CH2C=C / 1.08E-02 / 2.72E-09 / 1.00E-01
-2.72E-03 / 0.675 / Cholesterol (total) / 1.18E-02 / 4.18E-05 / 4.15E-05
-2.36E-03 / 1.015 / Cholesterol ester / 2.21E-02 / 7.65E-05 / 6.57E-05
2.21E-03 / 1.355 / (CH2)n / 8.00E-09 / 9.61E-08 / 3.13E-01
2.10E-03 / 1.305 / (CH2)n / 1.85E-02 / 4.29E-04 / 7.95E-01
-2.02E-03 / 1.995 / CH2C=C / 6.10E-03 / 1.09E-07 / 5.59E-01
-1.73E-03 / 0.855 / CH3 / 4.11E-01 / 4.16E-05 / 1.21E-05
-1.70E-03 / 1.285 / (CH2)n / 5.60E-03 / 2.21E-03 / 7.62E-02
1.69E-03 / 2.065 / CH2C=C / 1.86E-01 / 2.76E-05 / 1.60E-01
1.69E-03 / 2.765 / =CH-CH2-CH= / 2.86E-05 / 1.07E-05 / 6.00E-02
-1.67E-03 / 1.295 / (CH2)n / 3.69E-01 / 3.46E-02 / 8.55E-01
1.58E-03 / 0.895 / CH3 / 3.83E-04 / 1.37E-07 / 2.67E-03
1.57E-03 / 5.375 / CH=CH / 3.41E-06 / 7.49E-06 / 6.03E-02
1.54E-03 / 2.775 / =CH-CH2-CH= / 2.77E-05 / 5.62E-06 / 1.98E-02
1.46E-03 / 2.035 / CH2C=C / 3.53E-01 / 1.44E-08 / 1.73E-01
1.41E-03 / 1.345 / (CH2)n / 1.09E-06 / 3.34E-06 / 3.18E-02
1.38E-03 / 1.365 / (CH2)n / 1.50E-11 / 1.21E-11 / 4.54E-01
1.36E-03 / 5.365 / CH=CH / 1.87E-06 / 5.73E-06 / 7.16E-02

Supplemental Table S5:Loadings of IC1 of hydrophilic liver extracts *Confirmed by 2D ICA

Factor / Frequency / Compound / P-value
Control / NAFLD / P-value
Control / NASH / P-value
NAFLD / NASH
2.29E-02 / 3.265 / Betaine* / 2.04E-03 / 7.25E-04 / 4.32E-03
-9.27E-03 / 1.285 / ? / 3.45E-02 / 5.07E-02 / 5.58E-01
5.17E-03 / 3.905 / Betaine* / 2.11E-02 / 2.58E-03 / 4.44E-03
-5.11E-03 / 1.305 / ? / 7.86E-02 / 2.06E-01 / 2.69E-01
-4.82E-03 / 1.315 / ? / 5.85E-02 / 1.93E-01 / 2.09E-01
-4.36E-03 / 1.295 / ? / 3.53E-01 / 5.89E-01 / 5.05E-01
-3.68E-03 / 1.275 / ? / 4.81E-02 / 4.21E-02 / 9.91E-01
-2.18E-03 / 0.895 / ? / 3.42E-02 / 1.66E-01 / 1.18E-01
-2.03E-03 / 1.325 / Lactate / 3.34E-01 / 4.68E-01 / 6.40E-01
-1.52E-03 / 1.345 / Lactate / 5.94E-02 / 2.26E-01 / 1.72E-01
-1.51E-03 / 0.885 / ? / 2.79E-02 / 1.69E-01 / 3.79E-02
-1.46E-03 / 1.355 / ? / 3.66E-02 / 1.50E-01 / 1.37E-01
-1.44E-03 / 2.245 / ? / 2.79E-02 / 7.14E-02 / 1.96E-01
-1.41E-03 / 5.315 / ? / 2.53E-02 / 7.08E-02 / 1.44E-01
-1.40E-03 / 2.055 / ? / 2.96E-03 / 5.13E-03 / 5.37E-02
1.40E-03 / 3.275 / Taurine* / 1.31E-02 / 3.52E-01 / 1.93E-02
-1.26E-03 / 5.325 / ? / 3.64E-02 / 1.25E-01 / 2.44E-01
-1.19E-03 / 2.045 / ? / 4.48E-03 / 1.47E-02 / 1.69E-02
-1.17E-03 / 1.365 / ? / 2.54E-02 / 8.91E-02 / 1.63E-01
-1.11E-03 / 1.335 / Lactate / 5.46E-01 / 6.83E-01 / 7.83E-01
-1.09E-03 / 2.255 / ? / 4.02E-02 / 7.98E-02 / 3.73E-01
-1.09E-03 / 5.335 / ? / 3.03E-02 / 4.47E-02 / 7.22E-01
-1.08E-03 / 0.905 / Ile, Leu, Val / 7.34E-02 / 3.70E-01 / 1.51E-01
-1.03E-03 / 1.585 / ? / 3.09E-02 / 1.10E-01 / 1.46E-01
-9.51E-04 / 5.305 / ? / 3.04E-02 / 4.15E-02 / 5.90E-01

Supplemental Table S6: Loadings of IC2 of hydrophilic liver extracts. *Confirmed by 2D ICA

Factor / Frequency / Compound / P-value
Control / NAFLD / P-value
Control / NASH / P-value
NAFLD / NASH
1.04E-02 / 3.265 / Betaine* / 2.04E-03 / 7.25E-04 / 4.32E-03
-9.09E-03 / 3.425 / Taurine* / 2.71E-03 / 7.04E-01 / 2.92E-03
5.54E-03 / 1.285 / ? / 3.45E-02 / 5.07E-02 / 5.58E-01
-5.49E-03 / 3.415 / Taurine* / 2.47E-03 / 7.08E-01 / 2.82E-03
4.34E-03 / 1.305 / ? / 7.86E-02 / 2.06E-01 / 2.69E-01
4.20E-03 / 1.315 / ? / 5.85E-02 / 1.93E-01 / 2.09E-01
-4.10E-03 / 3.435 / Taurine* / 2.83E-03 / 7.85E-01 / 3.67E-03
-3.98E-03 / 3.255 / Betaine* / 1.70E-03 / 9.98E-01 / 5.45E-03
3.98E-03 / 1.295 / ? / 3.53E-01 / 5.89E-01 / 5.05E-01
3.59E-03 / 3.905 / Betaine* / 2.11E-02 / 2.58E-03 / 4.44E-03
-3.28E-03 / 3.245 / Betaine* / 5.42E-03 / 3.39E-01 / 2.05E-03
-3.10E-03 / 3.275 / Taurine* / 1.31E-02 / 3.52E-01 / 1.93E-02
2.16E-03 / 1.325 / Lactate / 3.34E-01 / 4.68E-01 / 6.40E-01
2.00E-03 / 0.895 / ? / 3.42E-02 / 1.66E-01 / 1.18E-01
1.88E-03 / 1.275 / ? / 4.81E-02 / 4.21E-02 / 9.91E-01
-1.66E-03 / 3.405 / Glucose* / 2.98E-02 / 8.82E-02 / 5.49E-03
1.54E-03 / 1.345 / Lactate / 5.94E-02 / 2.26E-01 / 1.72E-01
1.45E-03 / 0.885 / ? / 2.79E-02 / 1.69E-01 / 3.79E-02
-1.45E-03 / 3.835 / Glucose* / 1.87E-02 / 4.04E-01 / 7.02E-03
1.42E-03 / 1.335 / Lactate / 5.46E-01 / 6.83E-01 / 7.83E-01
-1.40E-03 / 3.475 / Glucose* / 2.92E-02 / 7.29E-02 / 5.64E-03
-1.37E-03 / 3.495 / Glucose* / 3.46E-02 / 5.48E-02 / 5.92E-03
-1.36E-03 / 3.845 / Glucose* / 1.65E-02 / 6.25E-01 / 9.10E-03
-1.36E-03 / 3.715 / ? / 2.12E-02 / 1.66E-01 / 6.14E-03
1.34E-03 / 1.355 / ? / 3.66E-02 / 1.50E-01 / 1.37E-01

Supplemental Table S7: Loadings of IC 5 for urine. *Confirmed by 2D ICA

Factor / Frequency / Compound / P-value
Control / NAFLD / P-value
Control / NASH / P-value
NAFLD / NASH
5.60E-03 / 3.435 / Taurine* / 1.25E-02 / 3.39E-04 / 9.94E-03
5.25E-03 / 3.265 / TMAO* / 6.43E-03 / 2.59E-03 / 3.11E-02
4.05E-03 / 3.275 / TMAO* / 8.76E-03 / 2.87E-03 / 3.14E-02
3.34E-03 / 3.425 / Taurine* / 9.51E-04 / 4.03E-05 / 1.16E-02
2.61E-03 / 3.445 / Taurine* / 1.77E-02 / 8.25E-04 / 1.24E-02
2.41E-03 / 3.255 / Taurine* / 2.90E-03 / 4.84E-05 / 1.90E-02
2.35E-03 / 2.895 / TMA* / 9.02E-03 / 2.29E-02 / 1.10E-01
-1.01E-03 / 4.055 / Creatinine* / 6.62E-02 / 3.32E-02 / 4.15E-01
9.08E-04 / 3.935 / Phosphocreatine* / 1.29E-01 / 2.20E-01 / 3.11E-02
-9.07E-04 / 3.045 / Creatinine* / 1.81E-01 / 1.65E-03 / 3.21E-02
-7.78E-04 / 3.735 / Ascorbate* / 1.36E-02 / 9.33E-08 / 7.20E-01
-7.31E-04 / 3.685 / ? / 8.73E-01 / 5.25E-04 / 3.26E-03
-7.19E-04 / 4.515 / Ascorbate* / 4.32E-03 / 6.16E-06 / 7.38E-01
-7.07E-04 / 3.745 / Ascorbate* / 9.03E-03 / 2.67E-05 / 4.79E-01
7.03E-04 / 3.035 / Phosphocreatine* / 2.03E-01 / 1.91E-01 / 5.91E-02
7.03E-04 / 2.695 / Citrate* / 3.92E-02 / 1.33E-02 / 4.12E-01
-6.98E-04 / 4.025 / Ascorbate* / 1.06E-02 / 5.84E-06 / 9.35E-01
-6.96E-04 / 1.245 / ? / 3.56E-05 / 7.38E-05 / 4.06E-02
5.95E-04 / 3.145 / Ethanolamine / 2.66E-01 / 3.49E-01 / 4.88E-01
-5.89E-04 / 3.675 / ? / 6.74E-01 / 6.89E-04 / 2.07E-02
-5.85E-04 / 1.235 / ? / 3.96E-05 / 1.68E-04 / 2.51E-02
5.62E-04 / 2.555 / Citrate* / 2.67E-02 / 5.23E-02 / 3.01E-01
-5.60E-04 / 3.775 / Ascorbate* / 1.61E-04 / 1.61E-04 / 4.70E-02
-5.56E-04 / 7.365 / Phenylalanine* / 6.78E-01 / 4.71E-03 / 1.79E-02
-5.25E-04 / 3.755 / Ascorbate* / 7.02E-03 / 1.76E-02 / 2.31E-01

Supplemental Table S8: Loadings of IC 4 for urine. *Confirmed by 2D ICA

Factor / Frequency / Compound / P-value
Control / NAFLD / P-value
Control / NASH / P-value
NAFLD / NASH
-8.88E-03 / 2.895 / TMA* / 9.02E-03 / 2.29E-02 / 1.10E-01
-4.04E-03 / 2.885 / TMA* / 1.95E-01 / 2.13E-01 / 1.57E-02
-4.04E-03 / 3.265 / TMAO* / 6.43E-03 / 2.59E-03 / 3.11E-02
1.81E-03 / 3.755 / Ascorbate* / 7.02E-03 / 1.76E-02 / 2.31E-01
-1.67E-03 / 3.415 / Taurine* / 3.01E-02 / 3.12E-02 / 9.53E-01
1.61E-03 / 3.045 / Creatinine* / 1.81E-01 / 1.65E-03 / 3.21E-02
-1.52E-03 / 2.575 / Citrate* / 1.49E-02 / 2.65E-02 / 6.09E-02
-1.44E-03 / 3.425 / Taurine* / 9.51E-04 / 4.03E-05 / 1.16E-02
-1.44E-03 / 2.685 / Citrate* / 4.39E-02 / 1.09E-01 / 1.46E-02
-1.43E-03 / 3.275 / Taurine* / 8.76E-03 / 2.87E-03 / 3.14E-02
1.29E-03 / 3.765 / Acorbate* / 5.72E-03 / 2.21E-02 / 8.07E-02
-1.19E-03 / 2.715 / Citrate* / 1.21E-01 / 1.40E-01 / 8.09E-01
-1.13E-03 / 3.435 / Taurine* / 1.25E-02 / 3.39E-04 / 9.94E-03
-9.99E-04 / 3.255 / Taurine* / 2.90E-03 / 4.84E-05 / 1.90E-02
9.69E-04 / 1.245 / ? / 3.56E-05 / 7.38E-05 / 4.06E-02
-8.89E-04 / 2.545 / Citrate* / 5.08E-02 / 6.81E-02 / 1.02E-01
-8.59E-04 / 3.445 / Taurine* / 1.77E-02 / 8.25E-04 / 1.24E-02
8.51E-04 / 1.235 / ? / 3.96E-05 / 1.68E-04 / 2.51E-02
7.70E-04 / 0.925 / Leucine* / 4.33E-02 / 8.07E-01 / 3.23E-03
7.42E-04 / 3.775 / Ascorbate* / 1.61E-04 / 1.61E-04 / 4.70E-02
6.85E-04 / 2.295 / ? / 6.32E-02 / 5.85E-01 / 2.07E-04
6.67E-04 / 4.065 / Creatinine* / 2.84E-01 / 1.82E-02 / 2.26E-01
6.51E-04 / 1.875 / ? / 2.53E-02 / 9.66E-01 / 1.91E-03
6.50E-04 / 2.305 / ? / 2.98E-02 / 1.59E-01 / 4.50E-03
-6.41E-04 / 2.725 / ? / 7.66E-03 / 1.69E-01 / 2.52E-02