Detection of Characteristic Phosphatidylcholine Containing Very Long Chain Fatty Acids in Cerebrospinal Fluid from Patients with X-Linked Adrenoleukodystrophy

Abstract

X-linked Adrenoleukodystrophy (X-ALD) is a rare genetic neurological disorder caused by a mutation of the ABCD1 gene that encodes a peroxisomal ABC protein ABCD1. ABCD1 has a role in transporting very long chain fatty acid (VLCFA)-CoA into the peroxisome for β-oxidation. ABCD1 dysfunction leads to reduced VLCFA β-oxidation and in turn increased VLCFA levels in the plasma and the cells of all tissues; these increased plasma levels have been used to diagnose X-ALD. It has been reported that plasma VLCFA is not correlated with the severity and disease phenotype of X-ALD. Therefore, we cannot predict the disease progression by the plasma VLCFA level. Cerebrospinal fluid (CSF) is constantly produced by brain, and thus levels of lipids containing VLCFA in CSF might be informative in terms of assessing X-ALD pathology. LC-MS/MS-based analysis showed that phosphatidylcholine (PC) containing VLCFA signals, such as PC 40 : 0(24 : 0/16 : 0), PC 42 : 0(26 : 0/16 : 0), PC 44 : 4(24 : 0/20 : 4) and PC 46 : 4(26 : 0/20 : 4) were characteristically detected only in the CSF from patients with X- ALD. In the present study, we analyzed limited number of patient’s CSF samples (2 patients with X-ALD) due to the limitations of the availability for CSF samples from this rare disease. However, our finding would offer helpful information for studying the disease progression biomarkers in X-ALD. To our knowledge, this is the first report of analyzing lipids containing VLCFA in CSF from patients with X-ALD.

Introduction

X-linked adrenoleukodystrophy (X-ALD) (OMIM300100) is one of the most common peroxisomal disorders with a minimal incidence of 1 in 17000 males.¹⁾ X-ALD is classified into two major clinical phenotypes: adrenomyeloneuropathy (AMN) and cerebral ALD (CALD). AMN occurs in almost all male adult patients in time and 80% of female adult patients presents slowly progressive myelopathy in the spinal cord.²⁾ CALD affects about 45% of male patients and is characterized by fatal cerebral inflammatory demyelination in the brain. All X-ALD patients have a loss-of-function mutation in the gene encoding ABCD1 that causes abnormal accumulation of very long chain fatty acids (VLCFA, defined as at least 22 carbons) such as hexacosanoic acid (C26 : 0) in various fluids and tissues including blood, brain and spinal cord.³⁾ Recently, measurement of lysophosphatidylcholine (LPC) 26 : 0 by LC-MS/MS of dried blood spots was used in newborn screening of X-ALD in several countries.⁴⁾ It has been reported that C26 : 0 plasma levels do not correlate with disease progression or disease severity. In addition, there is no clear genotype/phenotype correlation in X-ALD.²⁾ Therefore, a biomarker that could predict clinical course is urgently needed. Lipids containing VLCFA in plasma does not always reflect those levels in the brain, likely because of the blood–brain barrier.⁵⁾ Cerebrospinal fluid (CSF) contains many biomolecules that could contribute to the pathophysiology of X-ALD, including cytokines and lipids. Lund et al. reported that increased levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in CSF were correlated with X-ALD disease severity and could be used as early indicators for cerebral inflammation in CALD.⁶⁾ Therefore, levels of lipids containing VLCFA in the CSF would likely be informative because accumulation of VLCFA in the brain and spinal cord has been demonstrated to be an important pathological basis for X-ALD.⁷⁾ However, it has not been evaluated the lipids containing VLCFA in the CSF from patients with X-ALD. Theda et al. evaluated lipid composition of intact and active areas of demyelination in white matter of X-ALD patients and showed that the one of the most remarkable accumulation of VLCFA were observed in PC fraction, indicating that the increased levels of PC containing VLCFA might play a key role in the pathogenesis of X-ALD.⁸⁾ In addition, profiling of lipids in Abcd1-deficient mice brains revealed that one of the most increased classes of phospholipids containing VLCFA were PC.⁹⁾ Eichler et al. showed that injection of LPC containing VLCFA into the brain cortex caused microglial activation in mice, suggesting that LPC containing VLCFA may contribute to cerebral inflammation.¹⁰⁾ Therefore, certain kinds of PC and LPC containing VLCFA in CSF could be an informative biomolecule for the pathophysiology of patients with X-ALD.

In this study, we assessed levels of VLCFA-containing PC and LPC species in CSF from 2 patients with X-ALD and 3 healthy controls using LC-MS/MS.

We did not analyze a sufficient number of CSF samples from patients with X-ALD for statistical analysis due to the limitations of the availability for CSF samples from this rare disease. However, this study could provide useful information for a future study to find disease-related lipid molecules for X-ALD through discovering characteristic lipid molecules in CSF from patients with X-ALD.

Materials and Methods

Human CSF Samples

CSF from patients with X-ALD (n = 2) and healthy controls (n = 3) were obtained from the National Center for Neurology and Psychiatry BioBank (NCNP BioBank, Japan). Although we searched all the biobank that we could access in the world, but only those two CSF samples from patients with X-ALD were available. One male patient (X-ALD patient 1) in his thirties was diagnosed with AMN. This patient had pyramidal tract disorder and gait disorder. The other male patient (X-ALD patient 2) was in his fifties and was also diagnosed with AMN. This patient had gait disorder. All three healthy controls were male and one each was in their thirties, forties or fifties.

All procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients and all healthy controls who were enrolled in this study. The study was reviewed and approved by the Mitsubishi Tanabe Pharma Corporation review board (H-19-004).

Analysis of Lipids Levels by LC-MS/MS

To evaluate LPC levels, human CSF (100 µL) samples were homogenized in a mixture of 1-butanol and methanol. The homogenized samples were centrifuged, and the supernatants containing LPC were evaporated and then reconstituted in 100 µL methanol: formic acid (100 : 0.1, v/v) containing 100 nM d4-LPC 20 : 0, d4-LPC 22 : 0, d4-LPC 24 : 0 and d4-LPC 26 : 0. The LPC concentration was quantitated by tandem mass spectrometry (QTRAP6500 System (SCIEX) coupled with HPLC (Shimadzu Corp., Kyoto, Japan) using an internal standard method with deuterium labeled LPC containing the same acyl as the analyte. The mobile phase A consisted of water: formic acid (100 : 0.1, v/v) with 10 µM phosphoric acid and the mobile phase B contained acetonitrile: isopropanol: formic acid (50 : 50 : 0.1, v/v/v) with 10 µM phosphoric acid. Chromatography separation was performed on an ACQUITY UPLC BEH C8 column (2.1 × 100 mm, 1.7 µm; Waters, Milford, MA, U.S.A.) at 60 °C with a mobile phase B gradient of: 50% (at 0.00 to 0.50 min), 50 to 98% (at 0.50 to 4.00 min), 98% (at 4.00 to 6.50 min), and 50% (at 6.51 to 7.4 min); the flow rate was 0.4 mL/min.

The method for the analysis of PC was described in Supplementary Materials.

Results

To elucidate the profile of PC in CSF from patients with X-ALD, various PC species were analyzed by LC-MS/MS. We evaluated PC having an even number of carbons in the total acyl-chain length and detected 40 PC signals in the CSF samples (Supplementary file). The range of total carbon chain length in PC was 32–54. Among them, 4 signals (PC 40 : 0, PC 42 : 0, PC 44 : 4, PC 46 : 4) were detected in CSF from patients with X-ALD, whereas those peak areas were below the detection limit in healthy controls (Table 1). Notably, peak area of PC 46 : 4 in CSF from patients with X-ALD was at least 15 times higher than the detection limit. The acyl chain compositions of PC 40 : 0, PC 42 : 0, PC 44 : 4 and PC 46 : 4 were 24 : 0/16 : 0, 26 : 0/16 : 0, 24 : 0/20 : 4 and 26 : 0/20 : 4, respectively, indicating that these PCs containing C24 : 0 or C26 : 0 were characteristically existed in CSF from patients with X-ALD.

Table 1. Peak Areas of PC Detected Only in the CSF from Patients with X-ALD Using LC-MS/MS

Signals	Healthy control 1	Healthy control 2	Healthy control 3	X-ALD patient 1	X-ALD patient 2
PC 40 : 0 (24 : 0/16 : 0)	N.D.	N.D.	N.D.	1.5 × 106	1.1 × 106
PC 42 : 0 (26 : 0/16 : 0)	N.D.	N.D.	N.D.	1.1 × 106	3.2 × 105
PC 44 : 4 (24 : 0/20 : 4)	N.D.	N.D.	N.D.	5.7 × 106	1.4 × 106
PC 46 : 4 (26 : 0/20 : 4)	N.D.	N.D.	N.D.	4.8 × 106	5.5 × 106

CSF from patients with X-ALD and healthy controls were evaluated by LC-MS/MS. N.D. indicated the sample was below the detection limit (3.0 × 10⁵).

We also assessed the level of LPC because LPC 26 : 0 in plasma is used as reliable diagnostic marker in X-ALD.⁴⁾ Analysis of LPC by LC-MS/MS demonstrated that levels of LPC 26 : 0 in CSF from patients with X-ALD and healthy controls were 11.1–11.8 and 1.07–1.66 nmol/L, respectively (Table 2). In contrast, levels of LPC 20 : 0, 22 : 0 and 24 : 0 were comparable between CSF from patients with X-ALD and healthy controls.

Table 2. LPC Levels in X-ALD and Healthy Control CSF

	Healthy control 1	Healthy control 2	Healthy control 3	X-ALD patient 1	X-ALD patient 2
LPC 20 : 0 (nmol/L)	1.40	0.65	1.86	0.84	0.66
LPC 22 : 0 (nmol/L)	0.33	0.38	0.34	0.43	<0.30
LPC 24 : 0 (nmol/L)	4.27	2.77	3.21	7.47	2.64
LPC 26 : 0 (nmol/L)	1.66	1.07	1.46	11.8	11.1

CSF aliquots (100 µL) from patients with X-ALD and healthy controls were evaluated by LC-MS/MS.

Discussion

In X-ALD, VLCFA that accumulates in the CNS is thought to be largely derived from de novo biosynthesis due to the increase in fatty acid chain elongation activity and decrease in peroxisomal β-oxidation activity.¹¹⁾ CSF is constantly produced by the brain and is thereby a highly informative biofluid in terms of CNS pathology. Therefore, analysis of lipids containing VLCFA in CSF could give useful insight into the metabolic disturbance in the CNS of patients with X-ALD.

Using LC-MS/MS-based approach, we found that PCs containing VLCFA, such as PC 40 : 0(24 : 0/16 : 0), PC 42 : 0(26 : 0/16 : 0), PC 44 : 4(24 : 0 20 : 4) and PC 46 : 4(26 : 0, 20 : 4) were characteristically detected only in CSF from patients with X-ALD (Table 1). Large body of evidence showed that saturated VLCFAs including C24 : 0 and C26 : 0 were accumulated in CNS of patients with X-ALD.¹²⁾ In addition, the levels of PC containing VLCFA were increased in brain tissues from patients with X-ALD at autopsy, as compared to healthy controls.¹³⁾ It is thus reasonable that PCs containing saturated VLCFA, such as PC 40 : 0(24 : 0/16 : 0), PC 42 : 0(26 : 0/16 : 0), PC 44 : 4(24 : 0/20 : 4) and PC 46 : 4(26 : 0/20 : 4) were evidently existed in CSF from patients with X-ALD. In this study, the level of LPC 26 : 0 in CSF from two X-ALD patients appeared to be relatively high as compared with that of three healthy controls (Table 2). Therefore, further study is needed to confirm whether LPC 26 : 0 level is increased in CSF from patients with X-ALD.

Excess VLCFA has been suggested to be a cause of mitochondrial dysfunction, oxidative stress and endoplasmic reticulum (ER) stress, which may be a trigger for glial cell activation and lead to the inflammatory demyelination seen in X-ALD. It has been reported that the levels of PC containing VLCFA were higher in the active demyelination lesions when compared to the inactive states in the white matter of patients with X-ALD.⁸⁾ Together, PC and LPC containing VLCFA in CNS might play an important role in the pathogenesis of X-ALD.

Richmond et al. demonstrated that patients with X-ALD displayed higher levels of PC 44 : 4 in the plasma,¹⁴⁾ however it is unknown whether PC 44 : 4 is elevated in CSF. It is of interest that PC 44 : 4 was characteristically detected in CSF from patients with X-ALD in this study.

Future studies in a larger cohort of X-ALD patients CSF will be needed to determine if PC 40 : 0(24 : 0/16 : 0), PC 42 : 0(26 : 0/16 : 0), PC 44 : 4(24 : 0 20 : 4) and PC 46 : 4(26 : 0, 20 : 4) and LPC 26 : 0 levels in CSF correlate with disease severity and clinical course of X-ALD.

Acknowledgments

The authors thank Dr. Takashi Ono, Dr. Hideki Mochida and Dr. Rikako Yamauchi for their insightful comments and suggestions. The authors are grateful to NCNP BioBank, Japan for providing CSF samples from patients with X-ALD and healthy controls.

Conflict of Interest

In May 2021–March 2022, Masashi Morita acted as a consultant for Mitsubishi Tanabe Pharma Corporation, which funded this study.

Supplementary Materials

This article contains supplementary materials.

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