Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics
Abstract
:1. Introduction
2. Results
2.1. Patient Characteristics and NMR-Derived Metabolite Levels
2.2. Fatty Acid Oxidation: Steatosis vs. Fibrosis
2.3. Comparing Steatosis and Fibrosis Groups
2.4. Branched Chain Amino Acids (BCAA) and Aromatic Amino Acids (AAA)
2.5. Decreasing BCAA as a Function of Fibrosis Stage
2.6. TCA Cycle and Urea Cycle Metabolites: Steatosis
3. Discussion
3.1. Biobanked Sera of Bariatric Surgery Patients
3.2. Overview of Markers for NAFLD
4. Materials and Methods
4.1. Organic Extraction of Aqueous Metabolites
4.2. Serum Collection and Group Design
4.3. Targeted Profiling
4.4. Statistical Methodology and Data Analysis
4.5. NMR Data Acquisition
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameter | Non-NAFLD | Steatosis | Fibrosis | p (ANOVA) |
---|---|---|---|---|
Total, n | 32 | 39 | 29 | |
Sex | F (32) | F (39) | F (29) | |
Age (years) | 45 ± 11 | 45 ± 11 | 43.9 ± 8.9 | 0.832 |
Race and Ethnicity d | Caucasian (31) Native American (1) | Caucasian (38) African American (1) | Caucasian (29) | |
BMI (kg/m2) | 48.7 ± 6.9 | 50.1 ± 7.4 | 51.9 ± 8.0 | 0.268 |
Waist Circumference (inches) | 50.5 ± 4.4 | 52.8 ± 5.5 | 54.0 ± 5.1 | 0.021 |
(Waist/cm) | (128 ± 11) | (134 ± 14) | 137 ± 13 | |
Hypertension | 0.41 (n = 13) | 0.41 (n = 16) | 0.45 (n = 13) | 0.935 a |
Type 2 Diabetes | 0.41 (n = 13) | 0.38 (n = 15) | 0.48 (n = 14) | 0.713 a |
Hyperlipidemia | 0.41 (n = 13) | 0.31 (n = 12) | 0.41 (n = 12) | 0.632 a |
Blood sugar (mg/dL) | 98 ± 40 | 107 ± 55 | 122 ± 64 | 0.472 c |
Insulin (µU/mL) | 16 ± 11 | 26 ± 17 | 29 ± 16 | 0.003 b |
HbA1c (%) | 5.85 ± 0.65 | 6.3 ± 1.2 | 7.0 ± 1.8 | 0.005 b |
Triglycerides (mg/dL) | 150 ± 70 | 175 ± 92 | 280 ± 370 | 0.074 c |
Cholesterol (mg/dL) | 192 ± 37 | 192 ± 44 | 201 ± 60 | 0.686 |
High-Density Lipoproteins (mg/dL) | 53 ± 15 | 50 ± 11 | 48.0 ± 9.7 | 0.276 |
Low-Density Lipoproteins (mg/dL) | 110 ± 37 | 107 ± 43 | 108 ± 42 | 0.961 |
Alanine Aminotransferase (U/L) | 22.3 ± 9.1 | 28 ± 12 | 50 ± 35 | <0.001 b |
Aspartate Aminotransferase (U/L) | 23.8 ± 8.3 | 23.9 ± 6.8 | 42 ± 30 | 0.001b |
Lobular Inflammation (0/1/2/3) | 32/0/0/0 | 30/8/1/0 | 2/17/10/0 | |
Ballooning (0/1/2) | 32/0/0 | 30/5/4 | 4/13/12 | |
Steatosis Grading | ||||
0 (<5%) | 32 (100%) | 0 (0%) | 0 (0%) | |
1 (5–33%) | 0 (0%) | 26 (67%) | 0 (0%) | |
2 (34–66%) | 0 (0%) | 12 (31%) | 15 (52%) | |
3 (>66%) | 0 (0%) | 1 (3%) | 14 (48%) | |
Fibrosis Staging | ||||
0 (none) | 32 (100%) | 39 (100%) | 0 (0%) | |
1a (mild perisinusoidal) | 0 (0%) | 0 (0%) | 11 (38%) | |
1b (moderate perisinusoidal) | 0 (0%) | 0 (0%) | 2 (7%) | |
1c (portal/periportal) | 0 (0%) | 0 (0%) | 3 (10%) | |
2 (perisinusoidal and portal/periportal) | 0 (0%) | 0 (0%) | 8 (28%) | |
3 (bridging) | 0 (0%) | 0 (0%) | 5 (17%) | |
4 (cirrhosis) | 0 (0%) | 0 (0%) | 0 (0%) |
Metabolite | Non-NAFLD (n = 32) | Steatosis (n = 39) | Fibrosis (n = 29) | p (ANOVA) |
---|---|---|---|---|
2-Aminobutyrate | 0.0136 (66) | 0.0114 (42) | 0.0113 (40) | 0.208 a |
2-Hydroxybutyrate | 0.033 (22) | 0.026 (13) | 0.033 (14) | 0.158 b |
2-Hydroxyisovalerate | 0.0018 (13) | 0.0021 (13) | 0.0040 (42) | 0.005 b |
2-Oxoisocaproate | 0.0173 (64) | 0.0177 (46) | 0.0191 (64) | 0.462 b |
3-Hydroxybutyrate | 0.14 (13) | 0.076 (83) | 0.082 (93) | 0.066 c |
3-Hydroxyisobutyrate | 0.0057 (39) | 0.0048 (18) | 0.0057 (20) | 0.273 b |
2-Methyl-3-oxovalerate | 0.0109 (45) | 0.0117 (32) | 0.0132 (47) | 0.114 b |
Acetate | 0.033 (17) | 0.039 (65) | 0.0303 (61) | 0.569 c |
Acetoacetate # | 0.0087 (65) | 0.0071 (52) | 0.0071 (50) | 0.428 |
Acetone | 0.014 (13) | 0.0078 (53) | 0.0070 (60) | 0.003 b |
Alanine | 0.175 (43) | 0.209 (55) | 0.219 (61) | 0.003 |
Asparagine | 0.0195 (57) | 0.0201 (59) | 0.0181 (54) | 0.356 |
Aspartate | 0.0134 (61) | 0.0136 (61) | 0.0133 (52) | 0.964 |
Azelate * | 0.035 (24) | 0.032 (16) | 0.048 (42) | 0.086 b |
Betaine | 0.0198 (64) | 0.0208 (67) | 0.0167 (52) | 0.024 |
Carnitine | 0.0168 (41) | 0.0187 (45) | 0.0188 (65) | 0.154 a |
Choline | 0.0079 (24) | 0.0083 (22) | 0.0075 (22) | 0.328 b |
Citrate | 0.0186 (55) | 0.0179 (55) | 0.0199 (54) | 0.308 |
Creatine | 0.026 (37) | 0.0161 (67) | 0.0186 (97) | 0.445 c |
Creatinine | 0.027 (12) | 0.025 (12) | 0.023 (13) | 0.575 |
Dimethylsulfone | 0.05 (27) | 0.0036 (58) | 0.005 (13) | 0.937 c |
Formate | 0.0231 (39) | 0.0236 (34) | 0.0222 (30) | 0.258 |
Glucose | 1.88 (55) | 2.14 (75) | 2.4 (1.3) | 0.069 b |
Glutamate | 0.045 (17) | 0.050 (16) | 0.054 (21) | 0.180 |
Glutamine | 0.209 (41) | 0.207 (39) | 0.195 (50) | 0.410 |
Glycerol | 0.049 (23) | 0.050 (18) | 0.058 (25) | 0.163 |
Glycine | 0.131 (39) | 0.126 (27) | 0.126 (43) | 0.813 a |
Histidine | 0.0344 (56) | 0.0355 (59) | 0.0344 (71) | 0.696 b |
Hypoxanthine ** | 0.0059 (25) | 0.0060 (27) | 0.0042 (18) | 0.006 b |
Isobutyrate | 0.0056 (15) | 0.0053 (14) | 0.0057 (13) | 0.530 |
Isoleucine | 0.037 (18) | 0.0365 (70) | 0.0378 (86) | 0.900 b |
Lactate | 1.11 (43) | 1.25 (38) | 1.30 (40) | 0.171 b |
Leucine | 0.072 (30) | 0.067 (13) | 0.069 (15) | 0.641 b |
Lysine | 0.053 (14) | 0.057 (11) | 0.058 (13) | 0.259 |
Mannose | 0.0312 (61) | 0.033 (12) | 0.034 (12) | 0.565 b |
Methionine | 0.0135 (38) | 0.0137 (28) | 0.0132 (26) | 0.821 |
Ornithine | 0.026 (11) | 0.0260 (68) | 0.0244 (71) | 0.730 b |
Phenylalanine | 0.047 (14) | 0.047 (12) | 0.047 (10) | 0.985 |
Proline | 0.089 (36) | 0.098 (33) | 0.096 (40) | 0.534 b |
Propylene glycol | 0.088 (35) | 0.092 (29) | 0.117 (70) | 0.050 b |
Pyroglutamate | 0.0099 (45) | 0.0108 (39) | 0.0123 (54) | 0.115 |
Pyruvate | 0.0221 (93) | 0.031 (14) | 0.031 (17) | 0.015 b |
Serine | 0.045 (12) | 0.046 (11) | 0.045 (13) | 0.924 |
Suberate * | 0.035 (11) | 0.035 (10) | 0.042 (15) | 0.087a |
Taurine | 0.074 (40) | 0.070 (17) | 0.056 (13) | 0.031 b |
Threonine | 0.049 (16) | 0.053 (12) | 0.048 (14) | 0.232 |
Tryptophan | 0.0254 (72) | 0.0270 (63) | 0.0281 (50) | 0.242 b |
Tyrosine | 0.040 (13) | 0.0428 (91) | 0.044 (12) | 0.555 b |
Valine | 0.117 (38) | 0.118 (21) | 0.123 (26) | 0.689 b |
Non-NAFLD vs. Steatosis | Steatosis vs. Fibrosis | Non-NAFLD vs. Fibrosis | |||
---|---|---|---|---|---|
Metabolite (NMR) | p | Metabolite (NMR) | p | Metabolite (NMR) | p |
3-hydroxybutyrate * | 0.035 | 2-Hydroxybutyrate | 0.045 | 2-Hydroxyisovalerate | 0.010 |
Alanine | 0.004 | 2-Hydroxyisovalerate | 0.023 | Acetone | 0.007 |
Acetone | 0.012 | 3-Hydroxyisobutyrate | 0.046 | Alanine | 0.002 |
Pyruvate | 0.003 | Betaine | 0.008 | Betaine | 0.041 |
Hypoxanthine | 0.003 | Hypoxanthine # | 0.006 | ||
Suberate | 0.031 | Taurine | 0.025 | ||
Taurine | 0.001 | ||||
2-Hydroxybutyrate | 0.045 | ||||
Clinical | p | Clinical | p | Clinical | p |
Waist | 0.049 | ALT | 0.003 | Waist | 0.005 |
Insulin | 0.006 | AST | 0.003 | Insulin | 0.001 |
HbA1c | 0.046 | HbA1c | 0.002 | ||
ALT | 0.042 | Triglycerides * | 0.029 | ||
ALT | <0.001 | ||||
AST | 0.004 |
Fibrosis Stage 1 (n = 16) | Fibrosis Stages 2 and 3 (n = 13) | p Value | |
---|---|---|---|
Metabolite (NMR) | |||
2-Aminobutyrate | 0.0128 (41) | 0.0094 (32) | 0.021 |
Acetoacetate | 0.0087 (57) | 0.0053 (33) | 0.036 # |
Creatinine | 0.0295 (98) | 0.016 (13) | 0.001 # |
Hypoxanthine | 0.0051 (16) | 0.0032 (15) | 0.003 |
Isoleucine | 0.0422 (72) | 0.0324 (71) | 0.001 # |
Leucine | 0.076 (12) | 0.059 (14) | 0.001 |
Lysine | 0.063 (12) | 0.052 (11) | 0.020 |
Methionine | 0.0141 (26) | 0.0122 (21) | 0.040 |
Tyrosine * | 0.048 (13) | 0.0385 (85) | 0.050 # |
Valine | 0.135 (21) | 0.109 (24) | 0.004 |
Clinical | |||
Type 2 diabetes | 0.50 (8/16) | 0.46 (6/13) | 1.000 § |
Insulin | 31 (15) | 28 (17) | 0.475 # |
HbA1c | 6.5 (1.2) | 7.6 (2.3) | 0.232 # |
GFR + | 86 (20) | 90 (23) | 0.612 |
Variables | Non-NAFLD and Fibrosis (% Classification Success) | |||
---|---|---|---|---|
Overall | Non-NAFLD | Fibrosis | Nagelkirke R2 | |
Metabolite(NMR) only: | ||||
alanine, acetone, betaine, 2hiv | 85.2 | 90.6 | 79.3 | 0.478 |
betaine, hypoxanthine, tryptophan, taurine | 80.0 | 83.9 | 75.9 | 0.590 |
Metabolite(NMR) + clinical: | ||||
ALT, alanine, acetone, 2hiv | 86.9 | 87.5 | 86.2 | 0.732 |
ALT, insulin, propylene glycol, 2hiv | 86.2 | 89.7 | 82.8 | 0.726 |
Steatosis and Fibrosis (% Classification Success) | ||||
Overall | Steatosis | Fibrosis | Nagelkirke R2 | |
Metabolite(NMR) only: | ||||
pyroglutamate, betaine, taurine, 2hb | 85.3 | 89.7 | 79.3 | 0.507 |
azelate, hypoxanthine, taurine, 2hiv | 82.4 | 87.2 | 75.9 | 0.471 |
Metabolite(NMR) + clinical: | ||||
AST, taurine, azelate, 2hiv | 85.3 | 89.7 | 79.3 | 0.587 |
AST, taurine, glucose, 2hiv | 86.8 | 94.9 | 75.9 | 0.527 |
Non-NAFLD and Steatosis (% Classification Success) | ||||
Overall | Non-NAFLD | Steatosis | Nagelkirke R2 | |
Metabolite(NMR) only: | ||||
acetone, alanine, pyruvate, creatine | 73.2 | 65.6 | 79.5 | 0.314 |
acetone, alanine, pyruvate | 70.4 | 65.6 | 74.4 | 0.272 |
Metabolite(NMR) + clinical: | ||||
ALT, alanine, acetone, pyruvate | 76.1 | 68.8 | 82.1 | 0.358 |
AST, ALT, alanine | 78.6 | 77.4 | 79.5 | 0.305 |
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Robinson, E.J.; Taddeo, M.C.; Chu, X.; Shi, W.; Wood, C.; Still, C.; Rovnyak, V.G.; Rovnyak, D. Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics. Metabolites 2021, 11, 737. https://doi.org/10.3390/metabo11110737
Robinson EJ, Taddeo MC, Chu X, Shi W, Wood C, Still C, Rovnyak VG, Rovnyak D. Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics. Metabolites. 2021; 11(11):737. https://doi.org/10.3390/metabo11110737
Chicago/Turabian StyleRobinson, Emma J., Matthew C. Taddeo, Xin Chu, Weixing Shi, Craig Wood, Christopher Still, Virginia G. Rovnyak, and David Rovnyak. 2021. "Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics" Metabolites 11, no. 11: 737. https://doi.org/10.3390/metabo11110737