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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
Evolution of Metabonomics ?
Post-genomic Era of Biology
Genome Transcriptomics
Metabonomics
Genomics Gene expression Metabolism Proteins
Environmental stressors
Proteomics
dimethylglycine ?
acetate
2.62
zoom
Intensity
alanine
2.46
hypotaurine carnitine
arginine
2.8
2.6
1.6
We hypothesize that NMR-based metabonomics can provide evidence for the diagnosis of diseases, via the identification of metabolic profile.
Generate a hypothesis
Hypothesis-driven research
A rise in incidence due to cohort effect of hep B and C infections during 1980s.
In the year 2000, it was projected that there will be 430,000 deaths from HCC all over the world. The prevalence of HCC in an autopsy study among Indians were low, and varies from 0.2% to 1.9%.
Ref: Mohandas,K (2004)
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
46.7 36.2 28.2 22.1 9.4 6.5 3.0 3.9 3.1 2.7 2.5 2.2 2.1 2.7 1.8
Women
11.5 9.5 9.8 5.8 4.6 2.0 1.2 1.9 1.1 1.3 0.5 1.1 1.1 1.7 0.7
The mean age adjusted incidence of HCC in 6 Indian populations is 2.77 in males and 1.28 in females per 100,000 people.
Serology
liver function tests. AFP (alpha fetoprotein) blood test.
nodules > 2 cm, imaging techniques + appropriate serology can confidently establish the diagnosis without confirmation from a positive biopsy.
Ref: Bruix J et al, 2001.
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.
Sample size:
Keeping sensitivity of new test as 95 %, precision of population parameter as 10 %, and (1-) at 95%, the sample size was 18 positive cases.
Study setting
Gastroenterology department, Medical College, Trivandrum. (tertiary care centre)
NIST (National institute of science and technology) Pappanamcode, Trivandrum.
Study subjects
Cases consecutively admitted to Gastroenterology department in Medical College, with results of USG and appropriate serology tests (Inclusion criteria)
Exclusion criteria
Patients not giving consent. Patients without or not willing for further tests, for the confirmation of diagnosis as HCC or no HCC.
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 patients attending physician according to medical files.
ROC curve plotted & optimal cut off to discriminate HCC and no HCC
Area under the ROC curves - test's ability to discriminate between HCC and no HCC.
Combinations
Metabolome
Combinations in the metabolome which can improve accuracy of diagnosis of HCC will be summarized
Results
Baseline characters of total study subjects
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
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
0.748 0.753 0.857 0.719 0.743 0.802 0.722 0.818 0.820 0.756 0.808 0.792 0.767 0.714 0.700 0.704 0.732 0.818 0.811 0.710 0.768 0.846 0.701 0.739 0.703
0.001 0.001 0.000 0.005 0.002 0.000 0.004 0.000 0.000 0.001 0.000 0.000 0.001 0.006 0.010 0.008 0.003 0.000 0.000 0.007 0.001 0.000 0.009 0.002 0.009
0.623 0.621 0.765 0.589 0.615 0.691 0.595 0.705 0.717 0.634 0.705 0.678 0.648 0.573 0.557 0.573 0.604 0.714 0.707 0.567 0.643 0.755 0.561 0.611 0.559
0.873 0.885 0.949 0.848 0.870 0.914 0.849 0.930 0.923 0.878 0.912 0.905 0.886 0.854 0.843 0.836 0.860 0.921 0.916 0.853 0.892 0.936 0.842 0.866 0.847
51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73.
n-carbamoylaspartate n-carbamoyl--alanine o-phosphoserine pimelate proline propionate propylene glycol pyruvate sarcosine serine -alanine s-sulfocysteine suberate succinate succinylacetone sucrose tartrate threonate trans-4-hydroxy-l-proline trimethylamine trimethylamine n-oxide valine xylose
0.731 0.720 0.798 0.783 0.733 0.804 0.794 0.804 0.785 0.776 0.786 0.830 0.810 0.816 0.777 0.724 0.821 0.785 0.784 0.710 0.777 0.720 0.736
0.003 0.004 0.000 0.000 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.004 0.000 0.000 0.000 0.007 0.000 0.005 0.002
0.590 0.578 0.683 0.656 0.612 0.698 0.688 0.699 0.670 0.654 0.675 0.731 0.702 0.713 0.661 0.573 0.721 0.671 0.671 0.571 0.659 0.580 0.606
0.873 0.862 0.913 0.911 0.855 0.911 0.899 0.910 0.901 0.898 0.898 0.930 0.919 0.918 0.893 0.875 0.921 0.900 0.898 0.850 0.895 0.859 0.867
10. threonate 11. O-phosphoserine 12. 13. 14. 15. sarcosine propionate butanone citrate
16. tartrate 17. trimethylamine noxide 18. 3-hydroxy-3methylglutarate 19. glutamate 20. glutarate 21. s-sulfocysteine 22. ascorbate 23. 6-anhydro--dglucose 24. succinate 25. alanine
800.00 800.00 800.00 800.00 800.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00
700.83 700.83 680.75 660.67 540.17 750.00 720.92 720.92 720.92 700.83 700.83 700.83 700.83 700.83 700.83 680.75 660.67 600.42 830.33 790.17 750.00 720.92 720.92 700.83 680.75
20.74 00.28 20.74 00.28 20.56 00.29 20.40 00.30 10.75 00.37 30.00 00.33 20.77 00.34 20.77 00.34 20.77 00.34 20.57 00.35 20.57 00.35 20.57 00.35 20.57 00.35 20.57 00.35 20.57 00.35 20.40 00.36 20.25 00.37 10.89 00.41 40.20 00.36 30.36 00.38 20.80 00.40 20.58 00.41 20.58 00.41 20.40 00.42 20.24 00.44
29. 2-phosphoglycerate 330.9003 30. isobutyrate 31. pyruvate 32. pimelate 33. trans-4-hydroxy-lproline 34. valine 35. 2-aminoadipate 36. levulinate 37. propylene glycol 38. -alanine 39. suberate 40. succinylacetone 41. glycolate 42. xylose 43. glycylproline 44. 3-dihydroxyacetone 45. cystathionine 46. proline 47. 3-dimethylurate 48. 3-methylglutarate 50.2818 20.939 60.1551 120.8817 00.9707 90.8691 20.9556 80.2389 50.3824 90.5508 40.3623 40.4765 110.6971 110.6589 10.4628 80.0304 200.4469 20.1598 120.188
51. ethylene glycol 52. glutaric acid monomethyl ester 53. serine 54. 2-ethylacrylate 55. aspartate 56. 5-aminolevulinate 57. galactonate 58. homoserine 59. mannitol 60. trimethylamine 61. methylguanidine 62. guanidoacetate 63. methionine 64. n-dimethylglycine 65. isocitrate 66. methylsuccinate 67. galactitol 68. 2-oxoisocaproate 69. n-carbamoyl-alanine
10.9397 20.2698 160.8712 10.3727 100.4794 30.7126 60.0561 100.8915 60.0926 00.5 10.4669 40.1396 30.9777 00.8293 190.23 110.6588 20.4011 30.505 70.1585
700.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00 650.00 650.00 650.00 650.00 650.00 650.00 650.00 600.00 600.00
680.75 680.75 680.75 660.67 660.67 640.58 600.42 600.42 600.42 580.33 750.00 720.92 700.83 700.83 660.67 620.50 600.42 830.33 770.08
20.24 00.44 20.24 00.44 20.24 00.44 20.10 00.45 20.10 00.45 10.98 00.46 10.77 00.50 10.77 00.50 10.77 00.50 10.68 00.51 20.60 00.47 20.40 00.48 20.23 00.49 20.23 00.49 10.95 00.53 10.73 00.56 10.64 00.58 30.60 00.48 20.62 00.52
Improved sensitivity of many metabolites than the techniques in current practise. (Miller.J.C.et.al,2005)
Overall sensitivity and specificity of NMR based metabolome in the diagnosis of HCC have to be established.
Parallel combinations of metabolites can improve sensitivity. To improve the specificity, Different cut off Serial combinations (Consider the test as positive, if both metabolites are positive)
Recommendations
The Metabolome requires further validation with perfect gold standard. The cut offs suggested in this study is arbitrary (screening tool) and have to be changed, if using as a confirmatory test. Many metabolites like 2 butanone, is unique in hepatocellular carcinoma which is also reported in another recent study.
Known concentration of internal standards to improve the reliability. More precise estimates can be obtained if large molecular sized proteins are removed. Higher frequency NMR spectroscopy (>500 MHz), can give greater separation. NMR based metabonomics are cheaper (400Rs/sample) and faster (~ 5 min).
Conclusion
A Metabolome was identified for hepatocellular carcinoma.
Optimal cut offs to discriminate between hepatocellular carcinoma were determined. Many metabolites in the serum with high sensitivity and reasonable specificity which after validation can be used in routine clinical practise.
Acknowledgement
Express my gratitude towards one and every body, who directly & indirectly gave me their strength and wisdom to complete this thesis. I am deeply indebted to my supervisors, teachers and fellow trainees, for their helps, stimulating suggestions and encouragements
THANKING YOU