NMR based Metabolome to characterize and compare hepatocellular carcinoma with chronic liver disease and normal liver

Mr. Rakesh.N M.Phil trainee CERTC, Trivandrum

Metabonomics
Metabonomics is formally defined as, “The quantitative measurement of dynamic multi-parametric metabolic response of living systems to physiological stimuli or genetic modifications”. It summarizes the entire pool of metabolites in a bio-fluid, thereby promising a powerful diagnostic tool in future.
Metabolome or Metabolic profile
Ref: Nicholson et al., 1999

Metabonomics in health so far
• Diagnosis and classification of diseases (tumor types) • Time course disease progression • Learn pathological mechanisms • identify new biomarkers • Responses to treatment (efficiency, toxicity) • Drug design (decrease development time) • Generating databases (HMDB, tumor metabolome database)

Evolution of Metabonomics ?
Post-genomic Era of Biology
Genome Transcriptomics

Metabonomics
Genomics Gene expression Metabolism Proteins

Environmental stressors

Proteomics

liquid chromatography . LC.gas chromatography.Technologies commonly used in Metabolic profiling • NMR: Nuclear magnetic resonance spectroscopy • MS: Mass spectroscopy (coupled with GC or LC) • FTIR: Fourier transformed infra red spectra • CE: capillary electrophoresis GC.

Overview NMR-based metabonomic approach Tissue or biofluid sample NMR spectroscopy Measure the metabolite profile Multivariate analysis Treat profile as statistical ‘object’ for classification purposes Explore profile to gain mechanistic insight into the stress response 1H • Minimal sample preparation • Rapid analysis • Unbiased detector • Molecular structure .

58 2.62 zoom Intensity alanine 2.8 2.Sample spectrum with metabolic profiling Intensity 1-D 1H NMR spectra dimethylglycine ? acetate 2.46 hypotaurine carnitine arginine • extremely congested spectra (raw data) with hundreds of overlapping peaks 1.4 3 2.6 2.6 .2 2 1.50 Chemical shift (ppm) 2.8 Chemical shift (ppm) 1.54 2.4 2.

Generate a hypothesis Record limited dataset specific to that hypothesis Determine if hypothesis is true or false (next phase) Hypothesis-driven research .We hypothesize that NMR-based metabonomics can provide evidence for the diagnosis of diseases. via the identification of metabolic profile.

E.(2007) . Ref:Blum.H. called hepatocellular carcinoma 90% of all primary liver cancer is HCC 20 to 50% of patients presenting with hepatocellular carcinoma had previously undiagnosed cirrhosis.CANCER OF THE LIVER hepatocellular carcinoma Chronic liver cell injuries and regeneration stimulates a pathway of increased liver cell activation resulting in malignant transformation of hepatocytes.

Ref: Mohandas.2% to 1.9%.Global burden of HCC • Fifth most common malignancy in men & eighth in women worldwide • Age and male sex have a positive correlation • A rise in incidence due to cohort effect of hep B and C infections during 1980’s. and varies from 0. • The prevalence of HCC in an autopsy study among Indians were low.K (2004) .000 deaths from HCC all over the world. • In the year 2000. it was projected that there will be 430.

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0 3. .7 36.6 2.4 6.8 4.9 1.000 people.28 in females per 100.2 28.1 1.1 9.7 2.7 The mean age adjusted incidence of HCC in 6 Indian populations is 2.7 1.Age standardised incidence rate of HCC in different cities of India compared with rest of the world Place Japan (Osaka) Hong Kong China (Shanghai) Singapore Chinese Singapore Indians US SEER (Blacks) US SEER (Whites) Mumbai Trivandrum Bangalore Chennai Delhi Bhopal Karunagapally Barshi Men 46.5 9.1 2.5 3.8 5.1 1.5 9.2 2.2 1.77 in males and 1.5 1.8 Women 11.7 0.1 2.3 0.5 2.9 3.2 22.0 1.1 1.

wheat. soybeans. or too much iron in the liver (hemochromatosis) • Aflatoxins (from fungus that can contaminate peanuts.Etiology of Liver cancer • Hepatitis B (HBV) and hepatitis C (HCV) • Cirrhosis due to alcohol. and rice) • Tobacco use . hepatitis. groundnuts. corn.

. • CT scan or MRI scan of liver. • AFP (alpha fetoprotein) blood test.Diagnosis of hepatocellular carcinoma ? recommendations from EASL (European association for studies on liver) Serology • liver function tests. • Biopsy. Imaging • Ultrasound of the liver. • Angiogram of the liver. • Laparoscopy. • Blood tests for Hepatitis B and C.

Ref: Bruix J et al. • nodule size ≤ 2 cm . imaging techniques + appropriate serology can confidently establish the diagnosis without confirmation from a positive biopsy. .biopsy is recommended.According to EASL recommendations • The Gold standard in the diagnosis of liver cancer depends on the size of nodules. 2001. (imaging techniques do not have sufficient accuracy to distinguish HCC from other conditions & AFP levels usually remain within normal or slightly elevated) • nodules > 2 cm.

Rationale for Metabolic profiling in HCC .

which differentiates between these conditions . patients with cirrhotic liver (CLD) and apparently normal people from a basket of metabolome library. The secondary objective was to find optimal cutoff’s.Objective of the study To identify & characterise the distribution of significant metabolites in serum of patients with HCC.

.Methodology This study is a venture to adopt epidemiological principles into basic science research and is a sole attempt to highlight the use of Nuclear magnetic resonance (NMR) spectroscopy and Metabonomics principle in the diagnosis of hepatocellular carcinoma.

Research question Is there a different pattern of metabolite levels distributed in human serum samples analysed using 1H NMR based metabolome. which can distinguish patients with hepatocellular carcinoma from cirrhotic liver or people with apparently normal liver admitted to a tertiary care centre. .

. precision of population parameter as 10 %. the sample size was 18 positive cases. and (1-) at 95%.Study design: Descriptive study Sample size: Keeping sensitivity of new test as 95 %.

Trivandrum. . with results of USG and appropriate serology tests (Inclusion criteria) Exclusion criteria • Patients not giving consent. Trivandrum. • Patients without or not willing for further tests. (tertiary care centre) • NIST (National institute of science and technology) Pappanamcode. Medical College.Study setting • Gastroenterology department. for the confirmation of diagnosis as HCC or no HCC. Study subjects Cases consecutively admitted to Gastroenterology department in Medical College.

Reference Test  USG along with appropriate serology and expert opinions. (relatively imperfect gold standard when compared to histopathology)  approval from human ethical committee were obtained.  Data collection using Performa after informed consent  Performa was completed by patient’s attending physician according to medical files. .

• The serum samples were sent for NMR spectroscopy. • Tests for AFP and USG were measured on all patients. • CHILD Pugh score were recorded (as a summary index in progression of liver cirrhosis). chronic liver disease and normal liver. • The patients were grouped as Hepatocellular carcinoma. CT and MRI were used wherever required.Stepwise protocol in sample collection • Blood samples were collected and routine biochemical parameters were measured. .

Methodology for in vitro NMR spectroscopy • Sample collection. storage. loading into NMR tubes. • Metabolic profiles generated . 137 metabolites were selected) . • Preprocessing of spectrum in CHENOMX NMR SUITE. (250 metabolites considered. dilution with D2O. • Water suppression experiment. serum separation. • Spectrum was obtained from Bruker 400x NMR spectroscopy.

I (95%) Z test) Area under the ROC curves . (considered as 137 independent tests) 2x2 tables for each metabolite at different levels ROC curve plotted & optimal cut off to discriminate HCC and no HCC Sn & 1-Sp was calculated at different levels AUC was computed. .Data mining using ROC analysis 137 Metabolites.test's ability to discriminate between HCC and no HCC. (P-value and C.

if any one test is positive is considered as test positive) Combinations in the metabolome which can improve accuracy of diagnosis of HCC will be summarized . Sp.e. LR+ & LR-) Combinations Metabolome Parallel combinations of metabolites in metabolome (i..Metabolites with significant AUC & their Optimal cutoff’s Measures of accuracy (Sn.

Results Baseline characters of total study subjects .

Baseline characters of hepatocellular carcinoma group .

Experimental spectrum after processing .

26-11.19) 284.083) 5.950-3. 95% C.I) (Mean.18-91.I) 180 (134.068 (2.43) 58.36) 23.I) (Mean. 95% C.92) AST (IU/L) ALT (IU/L) ALP (IU/L) Bilirubin (mg/dl) CHILD-Pugh AST (aspartate transferase).68 (70.069) 11 (10.74) 104.02-225.43-385.60) 22.26 (183.98) 80.56) 0. 95% C.636 (1. ALT (alkaline transferase).900 (0.71-127.04-194.322) 8.04 (136.07 (80.717-1.03) 2.35 (20.Biochemical parameters of the total samples Characteristics HCC group CLD group Normal liver n=20 n=28 n= 20 (Mean.49-70.90-9.72-25.35 (19.93) 165.21 (45.75 (7.25 (55.28-5.98) 65.067-6.94-74. ALP(alkaline phospatase) .34-25.10) 4.60 (5.

593 0.865 0.003 0.661 0.754 0.872 0.639 0.911 .001 0. 3-hydroxy-3methylglutarate 14.807 p 95% Confidence value Interval 0. ascorbate 21. 2-hydroxyglutarate 7.000 0. 2-oxocaproate 10.001 0.760 0.894 0.763 0.878 0.700 0.664 0.835 0. 2-oxovalerate 12.881 0.001 0.775 0.000 0.828 0. citrate 24.927 0.589 0.734 0.000 0. 2-methylglutarate 8.750 0.Metabolome with significant Area under the ROC curve Sl Metabolites No 1.925 0.850 0. butanone 23.776 0.755 0.001 0.000 0.821 0.756 0.865 0.000 0.000 0.940 0. dimethylamine Area under curve 0.634 0.000 0.668 0.835 0.911 0. 2-oxobutyrate 9.930 0. 5-aminolevulinate 17.894 0.851 0.000 0.880 0. 2-ethylacrylate 6.583 0.628 0.659 0.000 0.650 0. alanine 20.000 0. 3-dihydroxyacetone 2.893 0.765 0. 2-oxoisocaproate 11.720 0.827 0. acetoacetate 18.729 0.699 0.004 0.704 0. 3-dimethylurate 3. aspartate 22. 2-aminoadipate 5. 2-phosphoglycerate 13.781 0.796 0. acetone 19.000 0.906 0.741 0.001 0. 3-methylglutarate 15.621 0.936 0.000 0.879 0.871 0.924 0.931 0.728 0.000 0.879 0.001 0.000 0.720 0. 4-hydroxybutyrate 16.922 0.622 0.747 0.001 0.000 0.890 0.731 0.779 0.809 0. 6-anhydro-ß-dglucose 4. cystathionine 25.660 0.724 0.805 0.005 0.644 0.695 0.803 0.

949 0.643 0. 32. 35.001 0.722 0. 42.008 0.573 0.808 0.589 0.756 0.886 0.009 0.892 0.26.557 0.811 0.705 0.005 0. 49.634 0.846 0.768 0.818 0.866 0. 29.615 0.717 0.001 0.559 0. 31.755 0.857 0.009 0.843 0. 33.004 0. 37.611 0.739 0.010 0.930 0.001 0. 30. 36.001 0.705 0.595 0.719 0.648 0.714 0. 45. 41.000 0.707 0.000 0.000 0.854 0.836 0.000 0. 27. 44.923 0.007 0.561 0. 50.621 0.567 0.703 0.921 0. 47.710 0.860 0.000 0.849 0. 28.905 0. 46.820 0.623 0.818 0.000 0.002 0.678 0. 39.802 0.853 0.878 0.003 0.573 0.936 0.870 0.753 0.743 0. 48.792 0.842 0.912 0. 38.002 0.700 0.732 0.604 0.848 0.916 0.006 0.873 0.701 0.748 0.001 0. 43. 34.767 0. ethylene glycol fructose galactarate galactitol galactonate glucarate glucose glutamate glutarate glutaric acid monomethyl ester glycine glycolate glycylproline guanidoacetate homoserine isobutyrate isocitrate levulinate malate mannitol methionine methylamine methylguanidine methylsuccinate n-dimethylglycine 0.885 0.691 0.000 0.000 0.000 0.704 0.914 0. 40.714 0.765 0.847 .

893 0. 61.573 0. 70.786 0. 60. 73.000 0. 66.855 0.698 0.590 0.661 0.000 0.785 0.000 0. 55.671 0.733 0.777 0. 57.776 0.699 0.000 0. 54. 53.003 0.850 0. 71.798 0. 64.721 0. n-carbamoylaspartate n-carbamoyl-ß-alanine o-phosphoserine pimelate proline propionate propylene glycol pyruvate sarcosine serine ß-alanine s-sulfocysteine suberate succinate succinylacetone sucrose tartrate threonate trans-4-hydroxy-l-proline trimethylamine trimethylamine n-oxide valine xylose 0.000 0.000 0. 52.873 0.000 0.702 0.810 0.910 0.659 0.731 0.919 0.794 0.720 0.571 0.656 0.654 0.000 0. 67.859 0.720 0.000 0.000 0.899 0.675 0.867 .901 0.862 0.783 0.000 0.731 0.804 0.913 0.612 0.784 0.000 0.895 0.804 0. 63. 72.004 0.898 0.688 0.777 0.578 0.713 0.875 0.898 0. 68.710 0.606 0.830 0.004 0. 56.670 0.51. 58.005 0. 59.000 0.816 0.921 0.821 0. 62.000 0.000 0.000 0.003 0.918 0.007 0.900 0.930 0. 69.002 0.736 0.911 0.724 0. 65.683 0.785 0.898 0.911 0.671 0.580 0.

9835 50.27 20.06 30.00 850.17 00.00 680. 13.8223 240.25 770.72 10.127 40.21 00.00 900.33 540.58 600.00 850.33 560.58 580. 5.83 640.75 640.5898 60.7368 50.7143 0.72 20.92 +LR 30.071 00.00 800.00 850.00 800. glutarate 21.40 30.077 00.54 20.4457 60.00 850. metabolites acetoacetate methylamine 2-oxovalerate dimethylamine 2-oxocaproate galactarate 2-hydroxyglutarate acetone glucarate Cut off’s (mM/L) 30. 2.00 900.75 680.14 20.08 750.00 950.27 10.3513 120.00 950.95 -LR 00.00 850.8257 00.8376 50.219 200.07 40.00 850.00 950.21 00.15 00.42 750. glutamate 20.60 20.0808 10.170 10. succinate 25.17 00.00 720.00 900.42 580.0864 100.13 00.00 800.13 00. 3-hydroxy-3methylglutarate 19. sarcosine propionate butanone citrate 16.75 680.25 850.75 680.15 00.88 20. 7.00 850. ascorbate 23.68 20.32 30. 14. tartrate 17.7027 120.75 560.92 720.092 00.15 00. threonate 11.27 00.22 00. 15.17 750.3481 130.00 850.18 00.5652 10.91 20.00 900. trimethylamine noxide 18. s-sulfocysteine 22.1926 40.14 30.00 720.6246 20.28 20.3998 60. 3.16 20. O-phosphoserine 12.7186 Sensitivit y 950.Metabolome and their measures of accuracy Sl no 1. alanine .19 00.086 00.00 900.88 20.33 580.21 00. 4.92 700.00 900.22 00.83 680.00 900.7171 20.94 50.23 00.00 900.8658 120.17 790.28 20.20 20.00 850.00 950.71 30.8376 40. 6.22 00.00 Specificity 700.26 20. 6-anhydro-ß-dglucose 24.72 20.27 00.20 00. 9.086 00.49 30.6427 30. 8.

8691 20. 2-phosphoglycerate 330.36 30.57 00.40 00.40 20.00 750.00 800.20 00.37 10.26.00 750.00 750.83 700.75 00. pimelate 33.5508 40.00 750. 2-methylglutarate 27. succinylacetone 41.00 750.57 00.00 720.80 00.24 00. n150. levulinate 37.00 700.34 20.67 600.83 680.35 20. trans-4-hydroxy-lproline 34.57 00.57 00.00 750.34 20.92 720.00 750.3909 30.342 carbamoylaspartate 50. suberate 40.00 700.35 20.00 750.00 750.2389 50.41 20.1551 120.9707 90.4628 80. glycine 28.77 00.33 20.58 00.44 29.89 00.92 720.35 20.74 00. pyruvate 32.77 00.35 20.17 750.36 20.00 800.00 700. 3-dimethylurate 48.83 700. 2-aminoadipate 36.28 20.00 700.41 20.00 700.30 10.42 20. ß-alanine 39.40 00. 3-dihydroxyacetone 45.00 700.92 720.42 830. isobutyrate 31.3615 .939 60.74 00.83 700. 4-hydroxybutyrate 60. valine 35.92 700.40 00.75 660.0304 200.58 00. glycolate 42.00 700. 3-methylglutarate 50.83 700.00 750.00 720.6971 110.83 700.34 20.35 20.188 49.00 750.00 800.77 00.17 750.57 00.3623 40.25 00.1598 120.67 540. malate 100. glycylproline 44. propylene glycol 38.00 00.38 20.6589 10.29 20.41 40.4469 20.57 00.56 00.00 750.4765 110.28 20.3824 90.00 750.33 790.75 20.75 660.83 680.9003 30.2818 20.8817 00.00 700. cystathionine 46.37 30.36 00.6442 140.83 700. xylose 43.9556 80.00 800.35 20.3447 800. proline 47.92 700.83 680.

00 700.73 00.42 600. galactitol 68. serine 54.2698 160.7126 60.10 00.00 700.48 20. mannitol 60. methylguanidine 62.00 700.83 700. glutaric acid monomethyl ester 53.98 00.64 00.00 650.00 700.75 680.1396 30.00 650.00 650.83 660.40 00.47 20.67 640.00 600.42 580.8915 60.49 10.1585 700.33 750.75 680.67 660.9397 20.4794 30.60 00.10 00.5 10.60 00.44 20.46 10.48 20. ethylene glycol 52.00 720.42 830.23 00.08 20. 2-ethylacrylate 55.00 680.44 20.56 10. aspartate 56.68 00.9777 00. 2-oxoisocaproate 69.24 00.00 650.51.24 00.62 00.50 10.23 110.00 600.77 00. isocitrate 66.58 600.00 700.75 660.95 00.50 10.00 700. n-dimethylglycine 65. n-carbamoyl-ßalanine 10.0926 00.4011 30.45 20.3727 100.51 20.6588 20.50 600.42 600.44 20.8293 190.8712 10.00 650.77 00.77 00. methionine 64.49 20.0561 100.4669 40.45 10. methylsuccinate 67.00 700. trimethylamine 61.52 .00 650. homoserine 59.92 700.53 10.33 770. galactonate 58.00 700.505 70.23 00.00 700.67 620. guanidoacetate 63.00 650.58 30. 5-aminolevulinate 57.50 10.24 00.

57 30.50 600.21 00.07 00.58 640.15 20.60 30.40 30.75 680.17 0 0 00.40 20.0 10.20 30.91 20.83 700.85 20.53 20.6-Anhydro-ßD-glucose 2 hydroxyglutarate and pimelate Pimelate and Cystathionine Glycylproline and Methylamine Methylamine and Galactarate Galactarate and Trimethylamine Noxide Alanine and Pimelate Acetone and cystathionine 1.55 20.20 00.10 .70 20.40 20.42 50 50 50 30.58 640.35 0 00.05 20.08 00.21 00.07 00.23 00.Combinations of metabolite to improve the diagnostic measures Sl No 1 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Metabolite Combinations Galactorate and Cystathionine Succinate and Cystathionine Pyruvate and Cystathionine 2-Hydroxyglutarate and Galactarate Butanone and pyruvate 3 hydroxy-3-methylglutarate and Succinate Cystathionine and Succinylacetone Serine and 2-Ethylacrylate Guanidoacetate and 1.13 00.06 00.6-Anhydro-ß-D-glucose and Ethylene glycol 2-methylglutarate and guanidoacetate Methylamine and Propionate 2-Oxocaproate and Methylamine Acetone and 2 ethylacrylate Sensi tivity 90 85 80 95 85 85 75 100 95 95 90 85 100 100 90 85 95 95 85 100 100 95 Specificity LR+ 75 75 75 700.82 20.08 00.83 700.83 680.90 LR00.54 20.11 00.91 20.0 20.75 660.58 620.26 20.67 660.67 640.27 00.67 660.82 20.83 700.42 600.6-Anhydro-ß-Dglucose 2-Methylglutarate and 1.15 00.23 0 0 00.20 30.58 640.

 Different cut off  Serial combinations (Consider the test as positive.al.O et. if both metabolites are positive) . • To improve the specificity. 2007) • Improved sensitivity of many metabolites than the techniques in current practise. (Arrieta. (Miller. • Parallel combinations of metabolites can improve sensitivity.Discussion of Results compared with AFP • An approximate comparison of sensitivity and specificity of metabolites with AFP.2005) • Overall sensitivity and specificity of NMR based metabolome in the diagnosis of HCC have to be established. as well as ultrasound can be made.et.C.J.al.

2 100 410.6 310.4 970.8 22 99 170.2 99 360.4 39 Sp 76 Sn 13 21 Sp 97 93 45 100 AFP 200 (ng/mL) Sn Sp AFP 400 (ng/mL) Sn Sp .6 950.3 32 100 0–64 100 65 41 60 87 94 900.2 980.3 100 80 80-94 18 200.9 470.Accuracy of AFP at different cut off’s (Literature search) Authors AFP 20 (ng/mL) AFP 100 (ng/mL) Sn Oka et al 1994 Pateron et al 1994 Peng et al 1999 Tong et al 2001 Trevisani et al 2001 Gebo et al 2002* Nguyen et al 2002 Gupta et al 2003* Farinati et al 2006 Arrieta et al 2007 60 63 41-65 54 600.1 990.

Recommendations • The Metabolome requires further validation with perfect gold standard. • Known concentration of internal standards to improve the reliability. is unique in hepatocellular carcinoma which is also reported in another recent study. . • Higher frequency NMR spectroscopy (>500 MHz). • More precise estimates can be obtained if large molecular sized proteins are removed. • The cut off’s suggested in this study is arbitrary (screening tool) and have to be changed. can give greater separation. • NMR based metabonomics are cheaper (400Rs/sample) and faster (~ 5 min). • Many metabolites like 2 butanone. if using as a confirmatory test.

Conclusion • A Metabolome was identified for hepatocellular carcinoma. . • Many metabolites in the serum with high sensitivity and reasonable specificity which after validation can be used in routine clinical practise. • Optimal cut off’s to discriminate between hepatocellular carcinoma were determined.

I am deeply indebted to my supervisors. stimulating suggestions and encouragements . who directly & indirectly gave me their strength and wisdom to complete this thesis. for their helps. teachers and fellow trainees.Acknowledgement Express my gratitude towards one and every body.

THANKING YOU .

USG.Diagnostic measures for AFP. & other imaging .

Comparison of Spectral images .

CHILD Pugh Score MELD Score (Model for end stage liver disease) .

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. • Sensitivity. which differentiates into HCC and no HCC were identified.70 was picked up for further analysis. specificity and likelihood ratios were computed for all metabolites individually at this cut off levels • Metabolites were combined parallel. • Area under the curve for each metabolite was calculated. above 0. • A suitable cut off for each metabolite.Specificity was computed at different levels. if any one test is positive is considered as test positive) improves the Sensitivity at minimal cost of Specificity. .Data mining using ROC analysis • ROC analysis were done for each metabolites (137 metabolites). (i.e. • Most significant AUC. (considering each one as independent test) • Sensitivity & 1.

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