Screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios

1. A method of identifying 27-hydroxylase (CYP27A1) deficiency in an animal, the method comprising:

receiving a dried blood spot comprising a blood sample from said animal;

enriching the blood sample by liquid chromatography, protein precipitation, liquid-liquid extraction, solid-liquid extraction, or ultrafiltration;

detecting, by mass analysis of the enriched blood sample, an intensity signal of at least a bile alcohol glucuronide and a C27- or C24-bile acid or a conjugate thereof, wherein the C27- or C24-bile acid or conjugate thereof is selected from the group consisting of:

cholic acid (CA), tauro-cholic acid (t-CA); glyco-cholic acid (g-CA); chenodeoxycholic acid (CDCA); tauro-chenodeoxycholic acid (t-CDCA); glyco-chenodeoxycholic acid (g-CDCA);

trihydroxycholestanoic acid (THCA); tauro-trihydroxycholestanoic acid (t-THCA); glyco-trihydroxycholestanoic acid (g-THCA); dihydroxycholestanoic acid (DHCA); tauro-dihydroxycholestanoic acid (t-DHCA); and glyco-dihydroxycholestanoic acid (g-DHCA);

calculating a ratio between the intensity signals of said bile alcohol glucuronide and said C24- or C27-bile acid or conjugate thereof;

comparing said ratio between the intensity signals of said bile alcohol glucuronide and said C24- or C27-bile acid or conjugate thereof with a ratio of a control sample or control value from a control animal of the same species as the animal but without CYP27A1 deficiency; and

identifying the animal as having hydroxylase (CYP27A1) deficiency, based on said comparison of said ratios.

2. The method according to claim 1 , wherein said bile alcohol glucuronide is cholestanetetrol glucuronide (tetrol).

3. The method according to claim 1 , wherein the intensity signal is determined of a stable isotope labeled compound selected from the group consisting of: bile alcohol glucuronide, a C24- or C27-bile acid, or a conjugate thereof, and wherein said intensity signal of said stable isotope labeled compound is compared to the intensity of said at least a bile alcohol glucuronide and a C24- or C27-bile acid or conjugate thereof.

4. The method according to claim 1 , wherein at least the intensity signal is determined of a C24-bile acid or conjugate thereof and of a C27-bile acid or conjugate thereof, wherein said C24-bile acid or conjugate thereof is selected from the group consisting of:

cholic acid (CA), tauro-cholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (t-CDCA), and glyco-chenodeoxycholic acid (g-CDCA),

and said C27-bile acid or conjugate thereof is selected from the group consisting of:

trihydroxycholestanoic acid (THCA), tauro-trihydroxycholestanoic acid (t-THCA), glyco-trihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), tauro-dihydroxycholestanoic acid (t-DHCA), and glyco-dihydroxycholestanoic acid (g-DHCA).

5. The method according to claim 1 , wherein said mass analysis is selected from the group consisting of: mass spectrometry (MS), High-resolution MS, liquid chromatography—mass spectrometry (LC-MS), matrix-assisted laser desorption/ionization—time of flight mass spectrometry (MALDI-TOF), quadrupole time of flight mass spectrometry (Q-TOF), Orbitrap-MS, and flow-injection MS.

6. The method according to claim 1 , wherein said animal is a human.

7. The method according to claim 1 , wherein said animal is a newborn.

8. The method according to claim 1 , further comprising measuring the intensity signal of said bile alcohol glucuronide or C24- or C27-bile acid or a conjugate thereof, relative to the intensity signal of an isotopically labeled standard.

9. The method according to claim 8 , wherein said isotopically labeled standard comprises a stable isotope labeled C24- or C27-bile acid or a conjugate thereof selected from the group consisting of:

cholic acid (CA), tauro-cholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (t-CDCA), glyco-chenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), tauro-trihydroxycholestanoic acid (t-THCA), glyco-trihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), tauro-dihydroxycholestanoic acid (t-DHCA), and glyco-dihydroxycholestanoic acid (g-DHCA).

10. The method of claim 9 , wherein said method comprises a step of ionizing the biological sample using a cone voltage of about 60 to about 90 V.

11. A kit comprising a stable isotope labeled C24- or C27-bile acid or a conjugate thereof selected from the group consisting of:

cholic acid (CA), tauro-cholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (t-CDCA), glyco-chenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), tau ro-trihydroxycholestanoic acid (t-THCA), glyco-trihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), tauro-dihydroxycholestanoic acid (t-DHCA), and glyco-dihydroxycholestanoic acid (g-DHCA); and

further comprising a positive control sample, wherein the positive control sample is a dried blood spot comprising a blood sample from a control animal of the same species as the animal but with cerebrotendinous xanthomatosis (CTX).