Bile acid biomarkers for Niemann-Pick diseases, methods and uses therefor
Methods for identification and quantification of bile acids are disclosed. Bile acids in plasma, serum and/or blood such as a dried blood spot are used to identify subjects with a Niemann-Pick disease. The methods include measuring levels of a bile acid, such as 3β,5α,6β-trihydroxycholanic acid, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)taurine, or a combination thereof. Detection of bile acids involve mass spectroscopy and/or a combination of mass spectroscopy and liquid chromatography such as a LC-MS/MS assay. The methods can be used with sphingomyelinase assays to detect, diagnose and differentiate between Niemann-Pick A/B and Niemann-Pick C (NPC) disease.
1. A method of detecting a Niemann-Pick disease in a subject comprising:
(a) providing a sample comprising plasma from a subject;
(b) extracting at least one bile acid from plasma by protein precipitation, resulting in an extracted sample, wherein the bile acid is a 3β,5α,6β-trihydroxy bile acid;
(c) adding a known amount of a synthetic internal standard comprising a stable isotope-labeled 3β,5α,6β-trihydroxy bile acid to the extracted sample;
(d) injecting the extracted sample comprising the synthetic internal standard into a liquid chromatography-mass spectrometer;
(e) measuring the concentration of at least one bile acid by calculating the amount of the at least one bile acid, wherein a peak area generated by the extracted sample is compared to a second peak area generated by the synthetic internal standard;
(f) comparing the amount of the at least one bile acid present in the sample to a reference value of the at least one bile acid obtained from a control population consisting of individuals not afflicted with a Niemann-Pick disease; and
(g) diagnosing the subject as afflicted with a Niemann-Pick disease if the amount of the at least one bile acid from the subject is statistically significantly greater than that of the reference value.
2. A method in accordance with claim 1 , wherein the Niemann-Pick disease is selected from the group consisting of Niemann-Pick A disease and Niemann-Pick B disease.
3. A method in accordance with claim 1 , wherein the Niemann-Pick disease is Niemann-Pick C disease.
4. A method in accordance with claim 1 , wherein the bile acid is selected from the group consisting of 3β,5α,6β-trihydroxycholanic acid (5α-cholanic acid-3β,5α,6β-triol), N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)taurine, and a combination thereof.
5. A method in accordance with claim 1 , wherein the at least one bile acid is 5α-cholanic acid-3β,5α,6β-triol N-(carboxymethyl)-amide.
6. A method in accordance with claim 1 , wherein the sample is a body fluid sample.
7. A method in accordance with claim 6 , wherein the body fluid sample is selected from the group consisting of a plasma sample, a serum sample, a blood sample, a sputum sample, a urine sample, an amniotic fluid sample, cord blood, and a combination thereof.
8. A method in accordance with claim 6 , further comprising adding a known amount of the synthetic internal standard to the body fluid sample.
9. A method in accordance with claim 1 , wherein a Niemann-Pick disease is diagnosed if the at least one bile acid concentration is at least 2 fold greater than the reference value.
10. A method for determining the status of a Niemann-Pick disease in a subject with a Niemann-Pick disease comprising:
(a) obtaining at least one body fluid sample comprising plasma from the subject;
(b) extracting at least one bile acid from plasma by protein precipitation resulting in an extracted sample, wherein the bile acid is a 3β,5α,6β-trihydroxy bile acid;
(c) adding a known amount of a synthetic internal standard comprising a stable isotope-labeled 3β,5α,6β-trihydroxy bile acid to the extracted sample;
(d) determining a quantity of 3β,5α,6β-trihydroxycholanic acid, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine, or N-(3β,5α,6β-trihydroxy-cholan-24-oyl)taurine by a liquid chromatography-mass spectroscopy analysis, wherein a peak area generated by the extracted sample comprising the synthetic internal standard is compared to a second peak area generated by the internal standard; and
(e) determining the magnitude of the difference between the quantity of 3β,5α,6β-trihydroxycholanic acid, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine, or N-(3β,5α,6β-trihydroxy-cholan-24-oyl)taurine and a reference value of the at least one bile acid obtained from the subject at an earlier time, wherein if the quantity of 3β,5α,6β-trihydroxycholanic acid, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine, or N-(3β,5α,6β-trihydroxy-cholan-24-oyl)taurine is statistically significantly greater than or equal to the reference value, then the subject is not deemed to exhibit remission of the Niemann-Pick disease.
11. A method of detecting a Niemann-Pick disease in a subject comprising:
(a) providing a sample comprising plasma from a subject;
(b) extracting at least one bile acid from plasma by protein precipitation, resulting in an extracted sample, wherein the bile acid is a 3β,5α,6β-trihydroxy bile acid;
(c) adding a known amount of a synthetic internal standard comprising a stable isotope-labeled 3β,5α,6β-trihydroxy bile acid to the extracted sample;
(d) injecting the extracted sample comprising the synthetic internal standard into a liquid chromatography-mass spectrometer;
(e) measuring the concentration of the at least one bile acid by calculating the amount of the at least one bile acid, wherein a peak area generated by the extracted sample is compared to a second peak area generated by the synthetic internal standard;
(f) comparing the amount of the at least one bile acid present in the sample to a reference value of the at least one bile acid obtained from a control population consisting of individuals not afflicted with a Niemann-Pick disease; and
(g) diagnosing the subject as afflicted with a Niemann-Pick disease if the amount of the at least one bile acid from the subject is statistically significantly greater than that of the reference value.
12. A method in accordance with claim 11 , wherein the Niemann-Pick disease is selected from the group consisting of Niemann-Pick A disease and Niemann-Pick B disease.
13. A method in accordance with claim 11 , wherein the Niemann-Pick disease is Niemann-Pick C disease.
14. A method in accordance with claim 11 , wherein the at least one bile acid is selected from the group consisting of 3β,5α,6β-trihydroxycholanic acid, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)taurine, and a combination thereof.
15. A method in accordance with claim 11 , wherein the at least one bile acid is 5α-cholanic acid-3β,5α,6β-triol N-(carboxymethyl)-amide.
16. A method in accordance with claim 11 , wherein the sample is a body fluid sample.
17. A method in accordance with claim 16 , wherein the body fluid sample is selected from the group consisting of a plasma sample, a serum sample, a blood sample, a sputum sample, a urine sample, an amniotic fluid sample, cord blood, and a combination thereof.
18. A method in accordance with claim 16 , further comprising adding a known amount of the synthetic internal standard to the body fluid sample.
19. A method in accordance with claim 16 , wherein a Niemann-Pick disease is diagnosed if the at least one bile acid concentration is at least 2 fold greater than the reference value.
20. A method in accordance with claim 16 , wherein the reference value is an average concentration of the at least one bile acid of a non-Niemann-Pick afflicted control group.
21. A method for determining the status of a Niemann-Pick disease in a subject with a Niemann-Pick disease comprising:
(a) obtaining a first body fluid sample comprising plasma from the subject;
(b) extracting at least one bile acid from plasma by protein precipitation, resulting in an extracted sample, wherein the bile acid is a 3β,5α,6β-trihydroxy bile acid;
(c) adding a known amount of a synthetic internal standard comprising a stable isotope-labeled 3β,5α, 6 β-trihydroxy bile acid to the extracted sample;
(d) comparing a peak area generated by the extracted sample to a second peak area generated by the synthetic internal standard, and calculating the amount of the at least one bile acid and determining a quantity of at least one bile acid selected from the group consisting of 3β,5α,6β-trihydroxycholanic acid, N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine, and N-(3β,5α,6β-trihydroxy-cholan-24-oyl)taurine by injecting the extracted sample comprising the synthetic internal standard into a liquid chromatography-mass spectrometer;
(e) administering a treatment for Niemann-Pick disease;
(f) obtaining a second body fluid sample from the subject;
(g) determining a quantity of the at least one bile acid in the second body fluid sample; and
(h) determining the magnitude of the difference between the quantity of at least one bile acid and between the first and second body fluid samples of the at least one bile acid.
22. A method in accordance with claim 1 , further comprising treating the subject with a compound selected from the group consisting of an iminosugar inhibitor of glucosylceramide synthase and 2-Hydroxypropyl-β-cyclodextrin.
23. A method in accordance with claim 1 , wherein the extracted sample is not chemically derivatized prior to injecting the extracted sample into the liquid chromatography-mass spectrometer.
24. A method in accordance with claim 10 , wherein the extracted sample is not chemically derivatized prior to the liquid chromatography-mass spectroscopy analysis.
25. A method in accordance with claim 11 , wherein the extracted sample is not chemically derivatized prior to injecting the extracted sample into the liquid chromatography-mass spectrometer.
26. A method in accordance with claim 21 , wherein the extracted sample is not chemically derivatized prior to injecting the extracted sample into the liquid chromatography-mass spectrometer.
27. The method of claim 1 , wherein the stable isotope is a non-naturally abundant C or N.
28. The method of claim 10 , wherein the stable isotope is a non-naturally abundant C or N.
29. The method of claim 11 , wherein the stable isotope is a non-naturally abundant C or N.
30. The method of claim 21 , wherein the stable isotope is a non-naturally abundant C or N.
31. The method of claim 1 , wherein the stable isotope is 15 N or 13 C.
32. The method of claim 10 , wherein the stable isotope is 15 N or 13 C.
33. The method of claim 11 , wherein the stable isotope is 15 N or 13 C.
34. The method of claim 21 , wherein the stable isotope is 15 N or 13 C.
35. The method of claim 1 , wherein the stable isotope is 13 C 2 , 15 N-glycine.
36. The method of claim 10 , wherein the stable isotope is 13 C 2 , 15 N-glycine.
37. The method of claim 11 , wherein the stable isotope is 13 C 2 , 15 N-glycine.
38. The method of claim 21 , wherein the stable isotope is 13 C 2 , 15 N-glycine.