A combination of 7-ketocholesterol, lysosphingomyelin and bile acid-408 to diagnose Niemann-Pick disease type C using LC-MS/MS

Abstract

Background: Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by mutations of NPC1 or NPC2, which encode the proteins that are responsible for intracellular cholesterol trafficking. Loss of this function results in the accumulation of cholesterol-related products, such as oxysterols, sphingolipids, and NPC-related bile acids, which were recently used as biochemical biomarkers for the diagnosis of NPC. Bile acid-408 is a new significant compound we found in Japanese NPC patients, and it likely belongs to the category of bile acids. However, the diagnosis of NPC using a single biomarker is not satisfactory for clinical application because of the high instance of false negatives or positives. Therefore, we proposed an application of NPC diagnosis using a combination of 7-ketocholesterol (7-KC), lysosphingomyelin (lysoSM), bile acid-408 and/or glucosylsphingosine (lysoGL-1).

Methods and findings: 7-KC, lysoSM and lysoGL-1 in sera and bile acid-408 in dried blood spots (DBS) were quantified within 17 minutes using tandem mass spectrometry and high-resolution mass spectrometry, respectively. We measured these biomarkers in NPC patients (n = 19), X-linked adrenoleukodystrophy (X-ALD) patients (n = 5), patients with other lysosomal diseases (n = 300), newborns (n = 124) and healthy people (n = 74). Our results showed a promising accuracy (97%) for NPC diagnosis using the combination of 7-KC, lysoSM and bile acid-408. However, contrary to our expectations, lysoGL-1 levels did not present at a significantly greater amount in NPC patients than other patients and negative controls.

Conclusions: The combination of 7-KC, lysoSM and bile acid-408 improves the accuracy of NPC diagnosis and is feasible for mass screening due to its simple sample preparation and measurement. Future research should investigate the chemical structure of bile acid-408 to further facilitate its advantage in diagnosis.

Fig 1. This is a plot of the amount of 7-KC in sera of NPC patients (n = 19), other patients (n = 295) and negative controls (n = 72).

One point is one sample. The error bar is mean value with standard deviation (SD). Red: 7-KC amount above 50 ng/mL among other patients or negative controls.

Fig 2. This is a plot of the amount of lysoSM in sera of NPC patients (n = 19), other patients (n = 300) and negative controls (n = 74).

One point is one sample. The error bar is mean value with standard deviation (SD). Red: lysoSM amount over 10 ng/mL among other patients; blue: lysoSM amount below the threshold in NPC patients.

Fig 3. This is a plot of the amount of bile acid-408 in NDBS (n = 124), DBS of NPC patients (n = 16) and negative controls (n = 67).

One point is one sample. The error bar is mean value with standard deviation (SD). Red: bile acid-408 amount over 2.4 ng/punch among negative controls; blue: bile acid-408 amount below the threshold in NPC patients.

Fig 4. These are the plots of correlations between biomarkers.

a: the correlation between 7-KC and lysoSM; b: the correlation between lysoSM and bile acid-408; c: the correlation between 7-KC and bile acid-408.

Fig 5. This is a plot of the combination of 7-KC, lysoSM and bile acid-408.

One point is one sample. Left: NPC patients (n = 16); middle: high risk suspicious (n = 12); right: negative controls (n = 38) with X-ALD patients in blue (n = 5). Two NPC patients (red) had only one abnormal biomarker.