403

ORIGINAL
Molecular species profiles of plasma ceramides in different
clinical types of X-linked adrenoleukodystrophy
Katsuya Morito1, Ryota Shimizu1, Hanif Ali2, Akina Shimada1, Tohru Miyazaki1, Naoko Takahashi1, M. Motiur Rahman1,
Kazuki Tsuji1, Nobuyuki Shimozawa3, Michiyasu Nakao4, Shigeki Sano4, Momoyo Azuma5, Meera Nanjundan6,7,
Kentaro Kogure1, and Tamotsu Tanaka1,6
1
Department of Pharmaceutical Health Chemistry, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8505,
Japan, 2Department of Medical Pharmacology, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8505, Japan,
3
Division of Genomics Research, Life Science Research Center, Gifu University, Gifu 501-1193, Japan, 4Depertment of Molecular Medicinal
Chemistry, Tokushima University Graduate School of Biomedical Sciences, Tokushima 770-8505, Japan, 5Department of Infection Control
and Prevention, Tokushima University Hospital, Tokushima 770-8503, Japan, 6Division of Bioscience and Bioindustry, Graduate School ofTechnology, Industrial and Social Sciences, Tokushima University, Tokushima 770-8513, Japan, 7Department of Cell Biology, Microbiology,
and Molecular Biology, University of South Florida, Tampa, Florida 33647, U.S.A.

Abstract : X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder associated with peroxisomal dysfunction. Patients with this rare disease accumulate very long-chain fatty acids (VLCFAs) in their bodies because of
impairment of peroxisomal VLCFA β-oxidation. Several clinical types of X-ALD, ranging from mild (axonopathy
in the spinal cord) to severe (cerebral demyelination), are known. However, the molecular basis for this phenotypic variability remains largely unknown. In this study, we determined plasma ceramide (CER) profile using
liquid chromatography-tandem mass spectrometry. We characterized the molecular species profile of CER in
the plasma of patients with mild (adrenomyeloneuropathy ; AMN) and severe (cerebral) X-ALD. Eleven X-ALD
patients (five cerebral, five AMN, and one carrier) and 10 healthy volunteers participated in this study. Elevationof C26 : 0 CER was found to be a common feature regardless of the clinical types. The level of C26 : 1 CER was
significantly higher in AMN but not in cerebral type, than that in healthy controls. The C26 : 1 CER level in the
cerebral type was significantly lower than that in the AMN type. These results suggest that a high level of C26 : 0
CER, along with a control level of C26 : 1 CER, is a characteristic feature of the cerebral type X-ALD. J. Med. Invest. 70 : 403-410, August, 2023
Keywords : ceramide, liquid chromatography-tandem mass spectrometry, X-linked adrenoleukodystrophy, peroxisomes

INTRODUCTION
X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder characterized by progressive demyelination in the central
and peripheral nervous systems and adrenal insufficiency (1, 2).
X-ALD is attributed to a mutation in ABCD1, which encodes an
ATP-binding cassette half-transporter (ALD protein) (3). Functional deficiency of ABCD1 results in the failure to transportvery long-chain fatty acids (VLCFAs) into peroxisomes, where
oxidative degradation of VLCFAs occurs. Consequently, thisdeficiency leads to the accumulation of VLCFAs, such as C24 : 0
FA and C26 : 0 FA, in the patient’s body (3). Because the ABCD1
gene is located on chromosome X, men with the mutated geneshow apparent symptoms of X-ALD from childhood to adulthood,
whereas half of female individuals with the mutated gene show
mild neurological symptoms in old age. In men, the prevalenceof X-ALD is estimated to be between 1 per 20,000–30,000 individuals (4).
Several clinical types of X-ALD, ranging from mild (axonopathy in the spinal cord) to severe (cerebral demyelination),
have been known (3). The clinical severity of X-ALD is unrelated to the genotype of ABCD1 mutation (3). Furthermore, theAbbreviations :AdolC, adolescent cerebral ; AMN, adrenomyeloneuropathy ; CC,
childhood cerebral ; CER, ceramide ; FA, fatty acid ; LC-MS / MS,
liquid chromatography-tandem mass spectrometry ; VLC, very longchain ; X-ALD, X-linked adrenoleukodystrophy.

correlation between the extent of VLCFA accumulation in apatient’s body and the clinical severity of X-ALD symptoms is notclear (5). In fact, there are patients with X-ALD with mild or noneurological symptoms despite elevated plasma VLCFA levels(6). At present, the mechanisms by which accumulated VLCFAs
are involved in the development of neurological degeneration in
X-ALD have not been elucidated, although VLCFA accumulation in the blood is known to be a robust diagnostic biomarker for
X-ALD (4). Furthermore, the molecular mechanisms underlying
phenotypic variability also remain unclear.
Ceramides (CERs) are intermediates of sphingolipid metabolism (7-9), and their N-acyl residues are predominantly occupied
by VLCFAs in mammalian cells including neural cells (10, 11).
CERs are biosynthesized from an acyl-CoA and a sphingoidbase by the enzymatic activity of six CER synthases, each ofwhich produces CERs with different N-acyl chain length (8).
Among them, CER synthase 2 which produces CERs containing
a VLCFA is highly expressed in many tissues including neuraltissues (12, 13). Thus, the VLCFA-accumulating condition incells of patients with X-ALD could cause the accumulation of
VLCFA-containing CERs in many cells.
Intracellularly produced CER plays a signaling role for growth
Received for publication April 11, 2023 ; accepted June 5, 2023.
Address correspondence and reprint requests to Tamotsu Tanaka, Division of Bioscience and Bioindustry, Graduate School of Technology,
Industrial and Social Sciences, Tokushima University, Tokushima
770-8513, Japan. E-mail : tanaka.tamotsu@tokushima-u.ac.jp

The Journal of Medical Investigation Vol. 70 2023

T. Tanaka, et al. Analysis of CER in X-ALD

404

arrest, differentiation, and apoptosis (14-19). Interestingly,
CERs of different chain lengths have different physiological andpathophysiological roles (20). In fact, studies that forcibly alter
the cellular CER composition by overexpression or silencing of
ceramide synthases show that long-chain fatty acid-containing
CERs are pro-apoptotic, while VLCFA-containing CERs are
anti-apoptotic (21-28). A part of them are also released outside ofcells as the component of lipoprotein particles (29) and exosomes(30, 31). The released CER-containing particles / vesicles can beuptaken by cells and affect their functions (32, 33). Considering
the chain length-specific role of CER, clarification of the molecular species profiles of CER in patients with X-ALD is worth
investigating to understand the pathology of X-ALD.
It has been shown that FA profile of plasma reflects thoseof body cells in X-ALD (34) to some extent. Based on this, the
VLCFA in plasma is used as a robust biomarker for diagnosis of
X-ALD (4). Here, we determined plasma CER levels using liquid
chromatography-tandem mass spectrometry (LC-MS / MS) andcharacterized the molecular profile of CER in the plasma of
patients with X-ALD with mild (AMN) and severe (cerebral)
symptoms. Our results suggest that a high level of C26 : 0 CER,
along with a control level of C26 : 1 CER, is a characteristic of thecerebral type of X-ALD.

extraction and analysis.
Plasma samples were collected from patients who underwent
X-ALD at the Graduate School of Medicine, Gifu University.
The study protocol was approved by the Ethics Committees of
the Graduate School of Medicine, Gifu University (No. 29-286)
and the Ethics Committees of Tokushima University Hospital(No. 2726). All methods were performed in accordance with theguidelines of the Declaration of Helsinki. Informed consent was
obtained from all participants or their legal guardians. Table
1 shows the sample size and mean age of patients with X-ALD
in each group as well as those of healthy volunteers. Patients’characteristics were summarized in the Supplementary Table 1.
Plasma from 11 patients with X-ALD and 10 healthy volunteers
were analyzed in a blinded manner, in which the specimen information was withheld from the investigator until the experiment
was completed.

Materials
N-lauroyl-D-erythro-sphingosine (C12 : 0 CER), N-palmitoyl-D-erythro-sphingosine (C16 : 0 CER), and N-lignoceroyl-D-erythro-sphingosine (C24 : 0 CER) were purchased from
Avanti Polar Lipids (Alabaster, AL, USA). CER from bovine
brain was obtained from Sigma-Aldrich (St. Louis, MO, USA).
All other reagents were of reagent, HPLC, or LC-MS grade.

Extraction of lipids from blood samples

PATIENTS AND METHODS

Collection of human blood samples
The study protocol, which involved healthy participants, wasapproved by the Ethics Committee of Tokushima University
Hospital (No. 2653). All methods were performed in accordancewith the guidelines of the Declaration of Helsinki. The influence of sex on CER concentration was evaluated using fasting
plasma samples from five men and three women. We found no
significant differences in the concentrations of each CER species
between groups. Thus, the analytical data were grouped without
sex discrimination.
To evaluate the influence of fasting, blood samples were collected from five healthy participants (four men and one woman)after overnight fasting. Non-fasting blood samples were collected
on another day from six healthy participants (four men and two
women) who consumed breakfast. To evaluate the influence ofsample preparation, fasting plasma and serum samples werecollected on separate days from the members in the same group
described above. It should be noted that there was no arbitrary
manipulation when we recruited participants for each experiment. Venous blood from healthy volunteers was transferred to a
blood collection tube containing EDTA-2K (NP-EK0405 ; Nipro
Medical Co., Ltd., Tokyo, Japan) for plasma preparation, or witha serum-separating agent (NP-SP1016 ; Nipro Medical Co.,
Ltd., Tokyo, Japan) for serum preparation. Tubes for plasma
preparation were immediately centrifuged (1,500 × g, 5 min, 3°C), while the serum preparation was incubated first for 30–60
min at room temperature before centrifugation. The supernatants of each tube were collected and stored at –80 °C until lipid

In a typical analysis, 0.1 mL plasma or serum was subjected
to lipid extraction using the Bligh and Dyer method (35) for
LC-MS / MS. Briefly, 0.1 mL of plasma or serum were mixed
with 1.0 mL of chloroform, 2.0 mL of methanol, and 0.7 mL of
KCl solution (40 mg / mL) on ice, and then sonicated for 10–20 s.
After adding 50 pmol of C12 : 0 CER as an internal standard,
1.0 mL each of chloroform and water were added to the mixture
for phase separation. After acidification of the aqueous layer to
pH 2–3 using 5 M HCl, the mixture was centrifuged at 1,100 ×g for 5 min. The chloroform layer was collected and the remaining upper layer was mixed with 2.0 mL of chloroform. Aftercentrifugation, the chloroform layer was collected again. The
combined chloroform layers were evaporated under a stream ofnitrogen. The recovered lipids were dissolved in 0.8 mL of methanol and then filtered through a non-polar filter (Chromatodisc
4 N, 0.2 μm, Kurabo, Osaka, Japan). The filtrate was evaporated again and the resultant lipids were dissolved in 0.1 mL of
methanol / formic acid (99 : 1, v / v) containing 5 mM ammonium
formate for LC-MS / MS analysis.

Analyses of CERs using LC-MS / MS
CER analyses were performed using a quadrupole-linear ion
trap hybrid mass spectrometer 4000 Q TRAP (Applied Biosystems / MDS Sciex, Concord, ONT, Canada) operating in multiple
reaction monitoring positive ionization mode with an Agilent
1100 liquid chromatograph (Agilent Technologies, Wilmington
DE, USA) and an HTSPAL autosampler (CTC Analytics AC,
Zwingen, Switzerland). CER species were detected using themultiple reaction monitoring mode and the detection condition of

Table 1. Sample size, mean age, and clinical type of each X-ALD patient group and healthy volunteers
Healthy
Male

X-ALD
FemaleCC(male)

AdolC(male)

AMN(male)

Carrier(female)

n

6

4

4

1

5

1

Age (years)

32.0 ± 11.4

24.3 ± 2.2

6.8 ± 1.3

14

50.6 ± 19.5

79

Age values are mean ± S.D. CC, childhood cerebral ; AdolC, adolescent cerebral ; AMN, adrenomyeloneuropathy.

The Journal of Medical Investigation Vol. 70 August 2023
each CER species is summarized in Table 2. Lipids were separated using a Cadenza CD-C18 column (50×2 mm, 3µm, Imtakt
Corp., Kyoto, Japan) at 42 °C and eluted with methanol / formic
acid (99 : 1, v / v) containing 5 mM ammonium formate as the mobile phase at an isocratic flow of 300 µL / min. The ratios of peakareas of objective CER species to internal standard (d18 : 1 / 12 : 0
CER) were used to determine the amount of each CER species.
Standard curves for correcting the ionization efficiency wereprepared by measuring the CER mixtures consisting of several
molar ratios of C12 : 0 CER and C16 : 0 or C24 : 0 CER (Fig. 1C).
Reciprocal values of slopes of standard curves prepared with
C16 : 0 (1.00) and C24 : 0 CER (1.47) were used as correction factors of ionization efficiency tentatively for long-chain (< C22) andvery long-chain (≥ C22) CER (VLC-CER), respectively.

Statistical analysis
Statistical analyses were performed using the Student’s t-test(to compare two groups) and one-way ANOVA with a post-hoc

405

Tukey test (to compare more than two groups). In all analyses, ap-value of < 0.05 was considered statistically significant.

RESULTS

Plasma or serum CER levels in healthy participants
The structures of the precursor and product ions selected for
CER detection using LC-MS / MS are shown in Fig. 1A. Retention times of each CER species measured in this study were
determined by analyzing CER mixture from bovine brain (datanot shown). Good separation of each CER species in humanplasma was achieved under these conditions (Fig. 1B). Standard
curves were prepared by measuring the CER mixtures consisting of several molar ratios of C12 : 0 CER and C16 : 0 or C24 : 0
CER (Fig. 1C). Peak area ratios of long-chain C16 : 0 and very
long-chain C24 : 0 CERs to those of the internal standard wereincreased lineally in a concentration-dependent manner.

Table 2. Detection condition of each CER species
CER species

Precursor ion[M + H]+

Product ion

DP (V)CE (V)

C12 : 0482.6

35

C16 : 0538.5

40

C18 : 0566.7

42

C20 : 0594.7

C22 : 0622.8

CXP (V)

45
264.4

40

48

C24 : 0650.9

C24 : 1648.6

50

C26 : 0

678.9

52.5

C26 : 1

676.9

52.5

16

50

DP, declustering potential ; CE, collision energy ; CXP, collision cell exit potential.

Fig 1. Determination of CER molecular species in human plasma using LC-MS/MS.

A) Structure of the precursor and product ions used for detecting CER. B) Plasma lipids from healthy participants were extracted and analyzed
using LC-MS / MS with C12 : 0 CER as an internal standard. Typical liquid chromatograms of CER in plasma are shown. C) Standard curves
were prepared using varying concentrations of C16 : 0 CER or C24 : 0 CER and fixed amount of C12 : 0 CER.

406

T. Tanaka, et al. Analysis of CER in X-ALD

We found that the dominant plasma CER species in humanparticipants were C24 : 0 CER (approximately 4.5 µM), followedby C24 : 1 CER (2.0 µM) and C22 : 0 CER (1.0 µM) (Fig. 2A). No
significant difference was observed in the levels of CER species
between overnight-fasted and non-fasted plasma samples. The
concentrations and compositions of plasma CER species in maleparticipants were similar to those in female participants (Fig.
2B). Furthermore, CER levels in the plasma and serum were
not significantly different (Fig. 2C), which agrees with previous
reports (36, 37).

Plasma CER levels in patients with X-ALDPatients with X-ALD exhibit an accumulation of VLCFA,
specifically C24 : 0 and C26 : 0 FAs, in their blood and tissues
because of genetic defect of transporting system of VLCFA into
the peroxisome (3). Fig. 3A shows the levels of CER species inindividual plasma samples from healthy controls (n = 10, open
columns) and patients with X-ALD (n = 11, filled columns). The
plasma concentrations of C24 : 0 (1.5-fold), C26 : 0 (4.3-fold),and C26 : 1 CERs (3.3-fold) in patients with X-ALD were significantly higher than those in healthy participants (Fig. 3B).
In contrast, the concentrations of C20 : 0 (0.66-fold), C22 : 0(0.67-fold), and C24 : 1 CERs (0.62-fold) in the plasma of patientswith X-ALD were slightly but significantly lower than those inhealthy controls. The ratio of C24 : 0 FA / C22 : 0 FA or C26 : 0
FA / C22 : 0 FA in total plasma lipids was used for the diagnosisof X-ALD (4). When we applied this methodology to the CER,
the variation of individual values in each group was reduced(Fig. 3C), and VLC-CER accumulation in patients with X-ALD

Fig 2. CER profiles in human plasma or serum.

became more apparent. The levels of C24 : 0, C26 : 0, and C26 : 1
CERs in patients with X-ALD were 2.4-, 6.3-, and 4.7-fold higher,
respectively, relative to those in healthy controls (Fig. 3D).
In an extended analysis, the patients were divided according to clinical severity (3) (Fig. 4). The childhood cerebral type(CC) and adolescent cerebral type (AdolC) are characterized byprogressive demyelination / neurodegeneration in the central nervous system. Here, we classified patients with CC and AdolC into
the “cerebral” (severe) type. Adrenomyeloneuropathy (AMN) is
characterized by relatively mild symptoms with neurodegeneration limited to the spinal cord. Patients with AMN and a female
carrier were classified into the “AMN” (mild) type. We observed
a significant increase in the level of C24 : 0 CER in patients with
AMN compared to that in healthy participants. The levels of
C26 : 0 CERs were significantly higher in both the cerebral and
AMN groups than in healthy participants (Fig. 4A). Analysis of
the data as values relative to C22 : 0 CER showed a significant
elevation in C24 : 0 CER and C26 : 0 CER levels in both typesof patients with X-ALD (Fig. 4B). In contrast, CERs with unsaturated VLCFAs such as C24 : 1 and C26 : 1 CER, seemed tochange in a clinical type-dependent manner. The C24 : 1 CERlevel of the cerebral group was significantly lower than that ofhealthy participants. The C26 : 1 CER level in the cerebral groupwas similar to that in healthy participants and significantly
lower than that in the AMN group (Fig. 4A). The tendency
unsaturated VLC-CER levels in the cerebral group are lower
than those in the AMN group is consistent with the findings of a
recent investigation conducted in the Netherlands (38).

Plasma or serum lipids from healthy participants were extracted and analyzed using LC-MS/MS. A) CER profiles in the plasma of healthy
participants with (n = 5) or without overnight fasting (n = 6). B) Plasma CER profiles in men (n = 7) and women (n = 3). C) CER profile in plasma
or serum (n = 5 for each group). Values are presented as the mean ± S.D.

The Journal of Medical Investigation Vol. 70 August 2023

407

Fig 3. CER profiles in plasma of patients with X-ALD and healthy controls.

Plasma lipids of patients with X-ALD and healthy controls were extracted and subjected to LC-MS/MS. A) CER concentrations in individual plasma
samples. n = 10 (healthy controls, open columns) and 11 (X-ALD, filled columns). B) Mean values of (A). C) Ratio of the concentration of each CER tothat of C22 : 0 CER. D) Mean values of (C). Values are presented as mean ± S.D. *p < 0.05, compared to healthy controls (Student’s t-test).

Fig 4. Comparison of plasma CER profiles of patients with X-ALD with different clinical severity.

A) Concentrations of CER species in the cerebral group, AMN group, and healthy controls. B) Ratio of concentration of each CER to that of
C22 : 0 CER in the cerebral group, AMN group, and healthy controls. Values are presented as mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.005 vs.
healthy controls and #p < 0.05 vs. the AMN group (one-way ANOVA with a post-hoc Tukey test).

408

T. Tanaka, et al. Analysis of CER in X-ALDDISCUSSION
In this study, we determined the molecular species profile of
CER in plasma and serum using LC-MS / MS. We found thatthe plasma CER profiles in male and female participants weresimilar, and that C24 : 0, C24 : 1, and C22 : 0 CERs were themajor molecular species in the plasma. We also found that theplasma and serum CER profiles were not significantly different,
as shown in previous studies (36, 37).
X-ALD is a demyelinating disease characterized by the systemic accumulation of VLCFA, particularly C24 : 0 and C26 : 0
FAs, due to the inability to import VLCFA into peroxisomes thatcarry out the oxidative degradation of VLCFA (3). The clinicalphenotypes of patients with X-ALD are highly variable and there
is no apparent correlation between the severity of X-ALD symptoms and the degree of VLCFA accumulation (3). We determined
the CER in the plasma of patients with mild (AMN) and severe(cerebral) X-ALD using the LC-MS / MS-based method developedin this study. We found that lower levels of unsaturated VLCCER, such as C24 : 1 CER and C26 : 1 CER, are features of cerebral X-ALD that distinguish it from AMN. This was unexpected,
because our recent study confirmed that unsaturated VLCFAs
with chain lengths greater than C20 are also peroxisomal β-oxidation substrates (39). Similar results have been observed inrecent European (38) and Korean (40) cohort studies. Richmond
et al. reported that a CER with an unsaturated VLCFA in theplasma was significantly lower in the cerebral group than in the
non-cerebral group (38). Lee et al. have shown that CER with
C26 : 1 in fibroblasts is considerably low in cerebral group ascompared to AMN group (40). Findings of these reports and our
results suggest that synthesis of unsaturated VLCFA-CER isdown-regulated in cerebral group, as hypothesized by Lee et al.
(40). Intriguingly, it has been reported that pharmacological induction of stearoyl-CoA desaturase 1, which produces C16 : 1 andC18 : 1 FAs from C16 : 0 and C18 : 0 FAs and thereby increases
levels of monounsaturated VLCFAs, reduces the toxicity of VLCFAs in fibroblasts derived from patients with X-ALD (41). The
accumulation of saturated VLC-CER without the accumulationof unsaturated VLC-CER may have a stressful effect on cellular
homeostasis in neuronal tissues.
In conclusion, elevation of C26 : 0 CER level was found to be
a common feature of X-ALD, regardless of the severity of theclinical type. C26 : 1 CER levels were significantly lower in the
cerebral group than in the AMN group. Although plasma CER
composition of healthy participants in this study is similar to
those in previous reports that analyzed CER composition inchildren (42, 43) and adults (44, 45), there is a limitation thatages of healthy controls were not matched completely to thosein patients with X-ALD. Small sample size in patients with
X-ALD is also the limitation in this study. Further studies witha larger number of patient samples as well as the age-matchedhealthy controls are needed to confirm clinical type-specific CER
profiles.

CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.

ACKNOWLEDGEMENTS
We thank the patients, their guardians, and healthy volunteers for donating blood for this study. This study waspartly supported by TR-SPRINT from the Japan Agency for
Medical Research and Development (AMED) (17lm0203008

and 18lm0203008 to TT) and the Research Program for Development of an Intelligent Tokushima Artificial Exosome (iTEX)
from Tokushima University.

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Supplementary Table 1. Summary of patients’ characteristics
Clinicalphenotype

Sex

Age(years)Adrenalinsufficiency

Administrationof Steroid

Administration ofLorenzo’s oil

CC

M

8

-

-

-

M

7

-

-

-

M

7

-

-

-

M

5

+ (high ACTH level)

-

-

AdolC

M

14

-

-

-

AMN

M

61

-

-

-

M

39

-

-

-

M

68

+

+

-

M

63

-

-

-

M

22

-

-

-

F

79

-

-

-

CarrierM, male ; F, female.