Differential diagnosis of liver disease
The present invention relates to the substantially non-invasive diagnosis of liver disease, especially to enable intervention in the progression of such disease at an early stage. This invention further relates to the use of plasma biomarkers to differentiate nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver (NAFL) and non-nonalcoholic fatty liver disease (NAFLD), and normal controls. Specifically, the invention relates to the use of free eicosanoids and other polyunsaturated fatty acid (PUFA) metabolite levels in plasma to differentiate NASH from NAFL and non-NAFLD normal controls.
1. A substantially non-invasive method of predicting or assessing the risk of progression of liver disease in a patient diagnosed with liver disease comprising:
directly treating a plasma sample from a subject with alcohol to dissolve free eicosanoids and free polyunsaturated fatty acid (fPUFA) to obtain free-dissolved eicosanoids and free-dissolved fPUFAs;
purifying the free-dissolved eicosanoids and free-dissolved fPUFAs;
measuring the level of one or more free eicosanoids and/or PUFA metabolites selected from the group consisting of (i) dhk PGD2, (ii) 20-COOH AA, (iii) 11,12-diHETrE and dhk PGD2; (iv) 11,12-diHETrE and 20-COOH AA; (v) dhk PGD2 and 20-COOH AA and (vi) dhk PGD2, 20-COOH AA and 11,12-diHETrE, and
determining the area under receiver operating characteristic curve (AUROC) based upon a ratio of the levels of the free-dissolved eicosanoids and/or free-dissolved fPUFA metabolites matched with deuterated internal standards of the same metabolite.
2. The method of claim 1 , wherein the liver disease is a nonalcoholic fatty liver disease (NAFLD).
3. The method of claim 2 , wherein the NAFLD is nonalcoholic steatohepatitis (NASH).
4. The method of claim 1 , wherein the ratio or a converted absolute value amount is communicated to a physician.
5. The method of claim 1 , wherein the one or more free eicosanoids and/or PUFA metabolites are dhk PGD2 and 20-COOH AA.
6. The method of claim 1 , wherein the one or more free eicosanoids and/or PUFA metabolites is 20-COOH AA.
7. The method of claim 1 , wherein the AUROC is about at least 0.8.
8. The method of claim 1 , wherein the AUROC is about at least 0.9.
9. The method of claim 1 , wherein the AUROC is about at least 0.99.
10. The method of claim 1 , wherein the risk of progression of liver disease is progression to cirrhosis.
11. A substantially non-invasive method of distinguishing nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver and non-nonalcoholic fatty liver disease (NAFLD) in a patient diagnosed with liver disease comprising:
directly treating a plasma sample from a subject with alcohol to dissolve free eicosanoids and free polyunsaturated fatty acid (fPUFA) to obtain free-dissolved eicosanoids and free-dissolved fPUFAs;
purifying the free-dissolved eicosanoids and free-dissolved fPUFAs;
measuring the level of one or more free-dissolved eicosanoids and/or free-dissolved fPUFA metabolites selected from the group consisting of (i) dhk PGD2, (ii) 20-COOH AA, (iii) 11,12-diHETrE and dhk PGD2; (iv) 11,12-diHETrE and 20-COOH AA; (v) dhk PGD2 and 20-COOH AA and (vi) dhk PGD2, 20-COOH AA and 11,12-diHETrE; and
determining the area under receiver operating characteristic curve (AUROC) based upon a ratio of the levels of the free-dissolved eicosanoids and/or free-dissolved fPUFA metabolites matched with deuterated internal standards of the same metabolite.
12. The method of claim 11 , wherein the one or more free eicosanoids and/or PUFA metabolites is dhk PGD2and 20-COOH AA.
13. The method of claim 11 , wherein the one or more free eicosanoids and/or PUFA metabolites is 20-COOH AA.
14. The method of claim 11 , wherein the AUROC is about at least 0.8.
15. The method of claim 11 , wherein the AUROC is about at least 0.9.
16. The method of claim 11 , wherein the AUROC is about at least 0.99.
17. A substantially non-invasive method of predicting or assessing the risk of progression of liver disease in a patient diagnosed with liver disease comprising
(a) obtaining a sample comprising one or more free eicosanoids and/or free-polyunsaturated fatty acid (fPUFA) metabolites from the plasma of a patient;
(b) spiking deuterated internal standards into each sample and primary standards used to generate a standard curve,
(c) treating the sample with alcohol to dissolve free eicosanoids and free polyunsaturated fatty acid (fPUFA) to obtain free-dissolved eicosanoids and free-dissolved fPUFAs,
(d) purifying the free-dissolved eicosanoids and free-dissolved fPUFAs;
(e) measuring the level of one or more free-dissolved eicosanoids and/or free-dissolved fPUFA metabolites selected from the group consisting of (i) dhk PGD2, (ii) 20-COOH AA, (iii) 11,12-diHETrE and dhk PGD2; (iv) 11,12-diHETrE and 20-COOH AA; (v) dhk PGD2 and 20-COOH AA and (vi) dhk PGD2, 20-COOH AA and 11 ,12-diHETrE,
(f) calculating the ratio between endogenous metabolite and matching deuterated internal standards;
(g) converting the ratios to absolute amounts by linear regression; and
(h) determining the area under receiver operating characteristic curve (AUROC) based upon the levels of the free-dissolved eicosanoids and/or free-dissolved fPUFA metabolites.