Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022

Review

Application of LC-ESI-MS/MS in the analysis of lipid species in samples
from patients with X-linked adrenoleukodystrophy, a peroxisomal disease
Kotaro Hama, Yuko Fujiwara, Kazuaki Yokoyama*
Faculty of Pharma-Sciences, Teikyo University, 2‒11‒1 Kaga, Itabashi-ku, Tokyo 173‒8605, Japan
Abstract

Peroxisomes are subcellular organelles that are involved in various biological processes, including lipid syn-thesis and metabolism. Inherited dysfunctions of enzymes in the peroxisome cause a number of peroxisomal disorders
associated with unusual lipid metabolites, as exempliﬁed by X-linked adrenoleukodystrophy (X-ALD), the most prevalent
peroxisomal disorder. Liquid chromatography linked to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is a pivotal technique for analysis of disease-speciﬁc metabolites to facilitate development of diagnostic markers andidentiﬁcation of disease-causing substances. Recent advances in MS have enabled intensive metabolomic targeting of verysmall amounts of lipids. However, it remains challenging to simultaneously analyze complex lipids such as glycosphingolipids. In addition, it is not easy to obtain stable isotopically labeled compounds for use as internal standards. Here, theapplication of LC-ESI-MS/MS in the analysis of phospholipids, glycosphingolipids (gangliosides, hexosylceramides, lactosylceramides, and globosides), and fatty acyl-coenzyme A is described. As an example, features of lipid species in X-ALD
are described on the basis of the authorsʼ previous studies. A novel method for preparation of deuterated free fatty acids isalso introduced, because deuterated compounds are useful as internal standards and probes for enzymes such as lysophospholipid acyltransferases that directly recognize a hydroxyoctadecadienoic acid (HODE) as an oxidized linoleic fatty acid.
Key words: phospholipid, glycosphingolipid, acyl-CoA, X-linked adrenoleukodystrophy, deuterated fatty acid
Abbreviations: ABCD1, ATP-binding cassette subfamily D1; ALD adrenoleukodystrophy; AMN, adrenomyeloneuropathy;acyl-CoA, fatty acyl-coenzyme A; CCALD, childhood cerebral ALD; CE, collision energy; DBS, dried blood
spot; GSL, glycosphingolipid; HIE, hydrogen-isotope exchange; HODE, hydroxyoctadecadienoic acid; HSCT,
hematopoietic stem cell transplantation; LC-ESI-MS/MS, liquid chromatography linked to electrospray ionization tandem mass spectrometry; lysoPC, lysophosphatidylcholine; SRM, selected reaction monitoring;
PEX, peroxin; PL, phospholipid; SED, single enzyme deﬁciency; VLCFA, very long-chain fatty acid; X-ALD,
X-linked ALD

Introductionoxidation of saturated or monounsaturated very long-chain
fatty acids (VLCFAs), α -oxidation of phytanic acid, andPeroxisomes are involved in biological processes such ashydrogen peroxide degradation and glyoxylic acid detoxiﬁ-

synthesis of plasmalogen, polyunsaturated fatty acids, andcation. A series of enzymes in peroxisomes is crucial for β -1)bile acids . Peroxisomal disorders are genetic disorders
caused by peroxisome dysfunction. The genes responsible* Corresponding author

for around 30 kinds of peroxisomal disorder have been
identiﬁed, although the causal relationships between aber-

Kazuaki Yokoyama
Faculty of Pharma-Sciences, Teikyo University, 2‒11‒1 Kaga,

rant metabolism due to dysfunctions of the genes and clini-

Itabashi-ku, Tokyo 173‒8605, Japan
E-mail: yokoyama@pharm.teikyo-u.ac.jp
Received: August 22, 2022.

2)

cal symptoms are still unclear in many of the disorders .
Therefore, it is critically important to identify the causative

Accepted: October 5, 2022.

molecules and/or metabolic machinery that explain thepathophysiology, which will also contribute to the develop-

Epub November 15, 2022.
DOI: 10.24508/mms.2022.11.006
101Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022‒

ment of diagnostic and therapeutic strategies for each peroxisomal disorder. Most peroxisomal disorders display spe-

5 8)these VLCFAs by β -oxidation . Consistently, patientswith X-ALD display an increased level of VLCFAs fromciﬁc lipid proﬁles that reﬂect the abnormal metabolic or. Thus, lipid

birth, and thus VLCFAs have been analyzed for the diagnosis of X-ALD. X-ALD is characterized by various clinicalanalysis represents an accurate and rapid diagnostic

phenotypes that diﬀer between patients and even between

approach to identifying peroxisomal disorders.

siblings with an identical genetic mutation in ABCD1 gene.
According to the severity and the onset period, X-ALD is

3,4)synthetic processes of lipids in peroxisomes

Liquid chromatography linked to electrospray ionization
tandem mass spectrometry (LC-ESI-MS/MS) enables struc-divided into subtypes, including childhood cerebral ALD

tural and quantitative analysis of a number of molecules
according to their mass-to-charge ratios (m/z) as well as(CCALD; the most common and severe phenotype, such as

analysis of their physicochemical properties such as hydrophobicity and molecular shape. In this review, the contribu-

and 10 years), adolescent cerebral ALD, adult cerebral

tion of LC-ESI-MS/MS to research into, and diagnosis of,noninﬂammatory distal axonopathy that develops after
puberty), and Addison only type (phenotype with symp-

demyelination of the brain at the age of onset between 3
ALD, adrenomyeloneuropathy (AMN; phenotype with aperoxisomal disorders is brieﬂy described. Technical issues
are also discussed in LC-ESI-MS/MS that the authors havetoms of adrenal insuﬃciency between 2 years and adult-

worked on for the analysis of X-linked adrenoleukodystrophy (X-ALD), the most prevalent peroxisomal disorder.

2)hood) . Importantly, there is not signiﬁcant correlation
between the rate of accumulation of VLCFAs and clinicalseverity, which makes it diﬃcult to provide a presymptom-

Pathology of Peroxisomal Disorders
Peroxisomal disorders are primarily classiﬁed into two

atic diagnosis and eﬀective treatment such as hematopoietic
stem cell transplantation (HSCT), which is the only cura-

categories: peroxisome biogenesis disorders, mainly caused

tive treatment and can prevent the progression of neurologi-

by mutations in peroxin (PEX) genes that are essential forcal symptoms .

9)

the assembly of peroxisomes (Table 1), and single enzyme

Current Application of MS for the Diagnosisof X-ALD

deﬁciencies (SEDs) caused by defects in enzymes that are2)

located and function in the peroxisome (Table 2) . New
peroxisomal disorders are still being identiﬁed by whole-ge-

Given the speciﬁc lipid proﬁles that reﬂect abnormalities

nome sequencing analysis.
X-ALD, a representative SED, is caused by dysfunctionin peroxisomal metabolism and the synthesis processes ofof ATP-binding cassette subfamily D1 (ABCD1). ABCD1

substantially to the diagnosis of peroxisomal disorders . In
particular, prompt diagnosis of X-ALD is essential because

lipids, conventional and accurate lipid analyses contribute10)

is located in the peroxisomal membrane and transports

early HSCT is crucial for improving the prognosis of
X-ALD patients. Traditionally, the analysis of VLCFAs in

VLCFAs with ≥ approximately 22 carbons from the cytosol
into the peroxisome, which leads to the degradation of
Table 1.

2)

Classiﬁcation of peroxisomal biogenesis disorders

DiseaseZellweger spectrum disorders
1
RCDP* type 1
1

RCDP* type 5
Broad phenotypes of PEX gene defects

Responsible gene
PEX1, 2, 3, 5, 6, 10, 12, 13, 14, 16, 19, 26
PEX7 (type 1)
PEX5-long isoform (type 5)

Mulibrey nanism

PEX1, 2, 3, 6, 7, 10, 12, 16
3
TRIM37*

PEX11 β deﬁciency
2
EMPF1*PEX11β4
DNM1 L*

Charcot-Marie-Tooth disease type 4A

GDAP1*

5*1 RCDP: Rhizomelic chondrodysplasia punctata. *2 EMPF1: Encephalopathy due to de3
fective mitochondrial and peroxisomal ﬁssion 1. * TRIM37: Tripartite motif containing
4
5
37. * DNM1 L: Dominant negative mutations in dynamin 1-like. * GDAP1: Ganglioside-induced diﬀerentiation-associated protein 1.

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Table 2.
Disease(responsible gene)
X-ALD(ABCD1)
1
ACOX1* deﬁciency

2)

Classiﬁcation of peroxisomal single enzyme deﬁciencies

Impaired biological/metabolic process

Substrate/product (function) of enzyme

Peroxisomal β -oxidation of fatty acids

VLCFA-CoA/(transporter)

Peroxisomal β -oxidation of fatty acids

VLCFA-CoA/enoyl-CoA(ACOX1)
2
DBP* deﬁciency
3(HSD17B4* )Peroxisomal β -oxidation of fatty acids

Enoyl-CoA/D-3-hydroxyacyl-CoA

4

Peroxisomal β -oxidation of fatty acids

3-ketoacyl-CoA/trimethyltridecanoyl-CoA, choloyl-CoA

Peroxisomal β -oxidation of fatty acids

SCPX* deﬁciency
5(SCP2* )6
AMACR* deﬁciency(AMACR)
7
ACBD5* deﬁciency

Peroxisomal β -oxidation of fatty acids(25R)-D/THC-CoA* , (2R)-pristanoyl-CoA/(25S)-D/THC-CoA,(2S)-pristanoyl-CoA
Acyl-CoA/(binding protein)(ACBD5)
8
ACOX2* deﬁciency

Bile acid synthesis(25S)-D/THC-CoA, (2S)-pristanoyl-CoA/enoyl-CoA

Bile acid synthesis

D/THC-CoA, phytanoyl-CoA, pristanoyl-CoA/(transporter)

11
BAAT* deﬁciency(BAAT)

Bile acid synthesis

Choloyl-CoA/taurocholate, glycocholate

Refsum disease
12(PHYH* )α -oxidation of fatty acids

Phytanoyl-CoA/pristanoyl-CoA

RCDP type 2
13(GNPAT* )Ether-phospholipid synthesis

21
DHAP* /acyl-DHAP

RCDP type 3
14(AGPS* )Ether-phospholipid synthesis

Acyl-DHAP/alkyl-DHAP

RCDP type 4
15(FAR1* )Ether-phospholipid synthesis

Acyl-CoA/fatty-alcohol

Acatalasemia
16(CAT* )

Hydrogen peroxide metabolism

Hydrogen peroxide/oxygen and water

Hyperoxaluria type1
17(AGXT* )18
GOX1* deﬁciency
19*(HAO1 )

Glyoxylate metabolism

Glyoxylic acid/glycine

Glyoxylate metabolism

Glycolic acid/glyoxylic acid

(ACOX2)
9
PMP70* deﬁciency
10*(ABCD3 )20*1 ACOX1: Acyl-CoA oxidase 1. *2 DBP: D-bifunctional protein. *3 HSD17B4: Hydroxysteroid 17-beta dehydrogenase 4. *4 SCPX: Sterol
567
carrier protein X. * SCP2: Sterol carrier protein 2. * AMACR: Alpha-methylacyl-CoA racemase. * ACBD5: Acyl-CoA binding domain
8
9
10
*
*
*
PMP70: Peroxisomal membrane protein 70 kDa.
ABCD3: ATP-binding cassette subcontaining 5. ACOX2: Acyl CoA oxidase 2.
11
12
13
*
*
BAAT: Bile acid CoA:amino acid N acyltransferase.
PHYH: Phytanoyl CoA 2-hydroxylase. * GNPAT: Glycefamily D member 3.
14
15
16
ronephosphate O-acyltransferase. * AGPS: Alkylglycerone phosphate synthase. * FAR1: Fatty acyl-CoA reductase 1. * CAT: Catalase.
17
18
19* AGXT: Alanine-glyoxylate and serine-pyruvate aminotransferase. * GOX1: Glycolate oxidase 1. * HAO1: Hydroxyacid oxidase 1.
*20 D/THC: Di- and tri-hydroxycholestanoyl-CoA. *21 DHAP: Dihydroxyacetone phosphate

X-ALD . C26:0-lysoPC is conventionally extracted fromsamples from X-ALD patients has been performed using
11‒13). More recently,
gas chromatography-mass spectrometry

15)

dried blood spot (DBS) samples using methanol anddirectly analyzed by LC-ESI-MS/MS. Thus, the analysis of

VLCFAs have also been analyzed by LC-ESI-MS/MS . In
14)

acids in a total lipid fraction are transformed into methyl

C26:0-lysoPC in DBS samples is suitable for high-through16,17). Large-scale screening of newput screening of X-ALD

ester forms, and the amount of C26:0 fatty acid and/or theborns for X-ALD is now conducted in the US (24 states andratio of C26:0 to C22:0 is used for the diagnosis of
4)
X-ALD . C26:0-lysophosphatidylcholine (lysoPC) was

the district of Columbia), the UK, and the Netherlands and18‒21),
has successfully identiﬁed novel lineages of X-ALD

also identiﬁed as a new marker for the diagnosis ofshowing the validity of LC-ESI-MS/MS for presymptom-

these approaches, both esteriﬁed and nonesteriﬁed fatty

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November 2022

channel in positive ion mode (Fig. 1A). For example, phos-

atic diagnosis. A newborn screening project targeting
X-ALD also started in 2021 in the Tokai Region of Japan,

phatidylcholine (PC) species containing either C16:0 andC18:1 (PC 16:0‒18:1) or C16:1 and C18:0 (PC 16:1‒18:0)

22)

conducted by Prof. Shimozawa of Gifu University .

can both be analyzed by an SRM channel targeting PC 34:1

Insight from Endogenous Lipids in Samples from Patients with X-ALD and ABCD1Deﬁcient Mice

(Fig. 1B). Note that phosphatidylinositol species, which canbe fragile at higher temperatures, were analyzed more sensitively in positive ion mode when the temperature of the

X-ALD is caused by the dysfunction of a single trans-

ESI source was set to 200°
C rather than 300°
C, while theporter, ABCD1, although the machineries that explain thesensitivity of other PLs was not signiﬁcantly inﬂuenced byphenotypic variance are still unclear, and even the causal

the temperature of the ESI source in the QTRAP4500 MS

relationships between the clinical phenotypes and VLCFAs
accumulated in X-ALD patients remain elusive. Most VLC-

instrument .

28)

FAs in X-ALD patients are not present in the free form but

After screening PL species that were signiﬁcantly alteredin samples from patients with X-ALD, the acyl moieties ofare incorporated into complex lipids such as phospholipids

PLs of interest were analyzed by product ion scanning in(PLs)

23,24). Hence, it is important to precisely analyze thenegative ion mode. MS/MS/MS analysis was performed
using a QTRAP4500 MS instrument for product ion scan-

endogenous forms of VLCFAs and clarify the abnormal
metabolic pathways caused by the dysfunction of ABCD1.
The application of LC-ESI-MS/MS-based techniques in thening because the ﬁrst and second precursor ions are tan-analysis of PLs, glycosphingolipids (GSLs), and fattyacyl-coenzyme A (acyl-CoA)-containing very long-chain(LIT), respectively. Then, accumulated second precursor

demly selected by quadrupoles (Q) 1 and 3/linear ion trap
ions are further subjected to collision induced dissociationin Q3/LIT, which improves the signal-to-noise ratio of thefatty acyl moieties is described below.

product ions (Fig. 1C). The structure of each PL speciesPLs

was determined from the product ions derived from two
fatty acyl moieties and at least one lysophospholipid (lys-Glycerophospholipids consist of a glycerol backbone
linked to polar headgroups at the sn-3 position and one oroPL) for glycerophospholipids as well as a fatty acyl-moi-

two acyl moieties at the sn-1 and/or -2 positions. Sphingomyelin contains phosphorylcholine and an acyl-moiety

ety and a demethylated sphingosylphosphorylcholine for
28,29). Notably, the positions of each fatty
sphingomyelins
acyl-moiety in the glycerol backbone can also be deter-

linked to the hydroxy and amino groups of a sphingoidbase, respectively. LC-ESI-MS/MS using a reverse phasemined by comparing the spectral intensities of two lysoPL
ions produced from precursor ions because the ion corre-columns such as an octadecyl (C18) column has been widely
applied to intensively analyze a number of glycerophospho-

sponding to 1-acyl-lysoPL arising from loss of the acyl-moiety at the sn-2 position is more abundant than the ion oflipids and sphingomyelin species with diﬀerent polar headhydrophobicities of PL species, samples were homogenized

2-acyl-lysoPL arising from loss of the acyl-moiety at the
30,31). As exempliﬁed by PC
sn-1 position (Fig. 1D)in methanol and total lipid fractions were extracted using

26:0/18:1 and PC 26:1/18:0, very long-chain saturated or

25,26)

groups and acyl moieties. Because of the wide range of27)

chloroform and methanol (Bligh and Dyer method) .

monounsaturated fatty acyl moieties were exclusively
located at the sn-1 position in the glycerol backbone in

Given that the total concentration of PL species harboring very long-chain fatty acyl moieties (VLCFA-PLs) ismost VLCFA-PL species in X-ALD ﬁbroblasts and28,32). These results are consistent with
abcd1-deﬁcient micesmall, the amount of PL species was ﬁrst quantiﬁed by
selected reaction monitoring (SRM, also called multiple
reaction monitoring, MRM), which has a high signal-to-previous reports on ﬁbroblasts from Zellweger spectrum

noise ratio. Advantageously, because each PL species disso-abnormally accumulated, indicating a molecular machinery

ciates into polar head and hydrophobic groups in this SRM

including enzymes that selectively recognize VLCFAs

disorder and a ﬁssion yeast mutant, in which VLCFAs were33,34).

analysis, multiple PL species with identical total carbon andGSLsdouble bond numbers in the acyl moieties as well as an

GSLs are composed of hydrophilic carbohydrate chains

identical polar head group can be detected by a single SRM
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Fig. 1.

Fragmentation pattern of phospholipids in positive ion mode.

(A) Phospholipid species dissociates into polar head and hydrophobic group in positive ion mode analysis. Generally, protonatedphosphorylcholine (m/z＝184) is mainly observed as the product ion of PC and SM species (marked as ʻaʼ and ʻcʼ, respectively). In
contrast, protonated hydrophobic groups were signiﬁcant and set as the quadrupole 3 (Q3) value of SRM channel (marked as ʻbʼ).
PC: phosphatidylcholine (X＝choline); PE: phosphatidylethanolamine (X＝ethanolamine); PS: phosphatidylserine (X＝serine); PI:
phosphatidylinositol (X＝inositol); PG: phosphatidylglycerol (X＝glycerol); SM: sphingomyelin.
(B) SRM channel settings for detection of a phospholipid molecular species and possible multiple assignments.
Multiple phospholipid species can be analyzed by a single SRM channel according to the total carbon and double bond numbers ineach phospholipid.
(C) Product ion of phospholipid in MS/MS/MS of negative ion mode.
The second precursor ion was produced by dissociating a portion of the polar head group of the negatively charged ﬁrst precursor
ion in Q2 and further fragmented into fatty acids (FA) (ʻeʼ and ʻgʼ) and lysophospholipid (ʻdʼ and ʻfʼ) in Q3/linear ion trap (LIT). The
lettering corresponds to those in Fig. 1D.
(D) Assignment of product ion spectra. In the case of PC 44:1, four spectra were assigned as two fatty acids and two demethylated
lysoPC species. Note that the intensity of the product ion spectrum from lysoPC 26:0 was greater than that from lysoPC 18:1. Thus,
the structure of PC 44:1 can be estimated as PC 26:0/18:1 rather than PC 18:1/26:0.

and a hydrophobic ceramide moiety. The metabolism of
GSL is associated with the pathogenesis of glycosphingo-

mide moieties, and thus GSLs harboring an identical cera-

lipidoses and is also implicated in X-ALD (Fig. 2A)

GM2, and GM3) cannot be easily separated .

35,36)

mide moiety with diﬀerent carbohydrate chains (e.g., GM1,
40).

Establishing methods that measure each molecular species
in GSL classes should aid pathophysiological characteriza-

To help overcome these problems, a novel LC-ESI-MS/
MS method using a chiral column (IF-3) that is usuallytion of GSL species, although the precise analysis of GSL

employed for the separation of structural isomers was

species has been challenging because of the remarkable

recently developed . In a series of experiments to optimize

diversity of GSLs. Separation on the normal phase or

the conditions, two parameters of the MS instrument werehydrophilic interaction chromatography columns that havefound to signiﬁcantly aﬀect the sensitivity of GSL analysis.

been used for the analyses of GSLs mainly depends on thesugar moieties of GSLs, and thus GSLs harboring an identi-

First, a higher temperature of the ESI ion source resulted inin-source fragmentation of GSLs and decreased sensitivity.

cal sugar moiety with diﬀerent ceramide moieties cannot be

For example, in positive ion mode using a QTRAP4500. In contrast, separation on

MS instrument, the signal intensities of three precursor ionsreversed-phase C18 columns mainly depends on the cera-of GT1b d18:1/C18:0 with diﬀerent adducts ([M＋2H] ＝37‒39)

eﬃciently separated

41)

2＋

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Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

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Fig. 2.

Fragmentation patterns and detected ions of GT1b d18:1/C18:0 in LC-MS/MS analysis.

(A) Structure of GT1b. Lettering (a‒f) marking the fragmentation patterns corresponds to the product ions listed in the table in (A)54)
and plot graph in (B‒G). The possible structure of fragment ions (f/f′) were indicated according to the previous study .
(B, C) Intensity of precursor ions (g, h, i, j) and fragment ions (a̶H2O, d, e) of GT1b d18:1/C18:0 at various ESI source temperatures in positive (B) and negative ion mode (C). Both precursor and in-source fragment ions were detected by Q1 scan analysis.
(D‒G) Representative patterns of product ions of GT1b d18:1/C18:0 generated through collision induced dissociation. Product2＋ions (a/a-H2O, b, c, d, e) derived from (i; [M＋2NH4] ) at collision energy (CE) of 30 V (D) and 90 V (E) in positive ion mode are2−marked. Similarly, product ions (a-H2O, d, f/fʼ/f”) from (j; [M−2H] ) at CE of −70 V (F) and −110 V (G) in negative ion mode aremarked. Both precursor and product ions were analyzed by enhanced product ion scan analysis. e: m/z＝264 was detected with larger CE in all GSLs analyzed.
2＋2＋1065.2, [M＋H＋NH4] ＝1073.6, and [M＋2NH4] ＝1082.2)
varied according to the temperature of the ESI, whereas

mode, by contrast, a precursor ion of GT1b d18:1/C18:0
2−([M-2H] ＝1063.2) was predominantly detected, while thefragment ions of a sialic acid (m/z＝292), a ceramide moiety

product ion of a sialic acid (m/z＝290) was detected even at(m/z＝548), and a sphingoid base (m/z＝264), which were
produced by in-source fragmentation, were signiﬁcant at a600°
C with a low (10 V) collision energy (CE) (Fig. 2C).

higher temperature of the ESI (Fig. 2B). In negative ion

GSL species. For example, in positive ion mode, structural

Second, the CE is crucial in the structural analysis of106Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022

and serve as metabolic intermediates of VLCFAs such as in
43,44)
β -oxidation and PL synthesis. Thus, it is important to

information on a ceramide (m/z＝548), a sialic acid (m/z＝274/292), and sugar chains (from m/z＝454 to 1837.7) waseﬃciently obtained at smaller CE of 30 V (Fig. 2D). In con-quantify the intracellular pool of each acyl-CoA species totrast, only a sphingoid long-chain base (m/z＝264) was eﬃ-

analyze the eﬀect of ABCD1 dysfunction on VLCFA
metabolism. However, when using conventional hydropho-

ciently obtained at larger CE of 90 V (Fig. 2E). In negative
ion mode, product ions from a sialic acid (m/z＝290), ceramide (m/z＝564), and sugar chains were signiﬁcant at

bic or hydrophilic interaction chromatography, the amphiphilic properties of acyl-CoA hamper the eﬃcient separa-

smaller CE of −70 V (Fig. 2F), while a sialic acid (m/z＝290) and an N-fatty acyl-moiety (m/z＝265/282/308) weretion of acyl-CoA species. Kasuya and co-workers
developed an LC-ESI-MS/MS method using ammonium

signiﬁcant at larger CE of −110 V (Fig. 2G). Note thatusing an accurate, high-resolution mass spectrometer, aacetate buﬀer (pH 5.3) and acetonitrile as the mobile phases
and analyzed short-, medium-, and long chain acyl-CoA

product ion spectrum from d20:1 sphingosine (calculated

speciesexact mass: m/z＝292.3004) was conﬁrmed to be signiﬁcant. Haynes, Merrill and co-workers used triethylamine as an ion-paring reagent in a mobile phase and devel-at higher CEs, while those from sialic acid (calculated exact
mass: m/z＝292.1033) were detected at lower CEs in posi-

oped an LC-ESI-MS/MS method to quantify long-chain47)
acyl-CoA species . Recently, acyl-CoA species were foundtive ion mode with a Q-Exactive MS instrument. This fea-

to be eﬃciently separated by LC-ESI-MS/MS using anture is useful for assigning the structures of GSL species.

octyl column with high sensitivity by adjusting the mobile

Following on from the above experiments and more
extensive experiments with GalCer, GlcCer, LacCer, Sul-phases to pH 9.0 with ammonium hydroxide, suggestingthat ion-paring reagents or ammonium hydroxide might befatide, GM3, GM2, GM1, GD1a, GD3, Gb3, and Gb4, a

crucial to attenuate interactions between acyl-CoA and the
HPLC column, such as chelate formation with residual sila-

45,46)

common fragment ion from d18:1 sphingosine (m/z＝264)
was applied as a product ion in each SRM channel with highnol groups and/or metals, for the eﬃcient separation ofacyl-CoA species. In the recent study, cells were homoge-

CE (＞70 V) in positive ion mode. Given that GSL species

nized in acetonitrile/isopropanol (3 : 1 by volume) and ana-

harboring a d18:1 sphingosine moiety make up the majorityof the total GSL fraction, the present method enables simul-

lytes were prepared by solid phase extraction using a
2-(2-pyridyl)ethyl silica gel column. Then acyl-CoA species

taneous measurement of a number of GSL species with highwere precisely analyzed by LC-ESI-MS/MS using deuterated C16:0-d31-CoA chemically synthesized from C16:0-d31

sensitivity. Cells were homogenized and deproteinized bymethanol, and analytes were measured using the
LC-ESI-MS/MS method and applying the chiral column (IF-free fatty acid (FFA C16:0-d31) as an internal standard (IS)compound. Hexacosenoyl (C26:1)-CoA, rather than hex-

3) and MS conditions described above. The level of C25- orC26-containing GSL species was found to be elevated inacosanoyl (C26:0)-CoA, was found to be most abundantamong the VLCFA-CoA species in ﬁbroblasts from patientsﬁbroblasts from patients with X-ALD. Notably, C44:1 HexCer (galactosylceramide and/or glucosylceramide) was sig-

32)
with X-ALD and ABCD1-deﬁcient HeLa cells . In con-niﬁcantly more accumulated in ﬁbroblasts from patientswith severe CCALD than in that with moderate-phenotype

trast, the amount of FFA C26:0 was larger than the amountof FFA C26:1 in ﬁbroblasts from patients with X-ALD

AMN, indicating diﬀerent proﬁles of GSL species containing VLCFAs between patients with diﬀerent clinical pheno-(unpublished results), suggesting that intracellular C26:0CoA is more eﬃciently metabolized than C26:1-CoA.

42)

types . Further precise analysis of GSL species will aid in
discriminating between diﬀerent X-ALD subtypes.

Given that chloroquine was recently identiﬁed to attenuateabnormal behavior in ABCD1-deﬁcient zebraﬁsh by

Acyl-CoA

increasing the expression level of stearoyl-CoA desaturase-1, it would be fascinating to determine whether a meta-

Acyl-CoA species are amphiphilic metabolites composedof a hydrophilic CoA and a hydrophobic fatty acyl-moiety

bolic diﬀerence between saturated and monounsaturated
48)
VLCFA-CoAs is involved in the pathology of X-ALD .

joined by a thioester linkage. Very long-chain fatty acyl-coenzyme A (VLCFA-CoA) species are endogenously synthesized through the fatty acid elongation process by ELOVL1
107

Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

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Application of Deuterium-labeled Fatty Acids
in Lipidomic Analysis

hydroxylated fatty acids as starting materials, providing a

Stable isotopically labeled analogs are desirable as IS
compounds because these unnatural compounds can prop-

analysis that have previously been diﬃcult to prepare. In
particular, the α -deuterium of the tetradeuterated fatty

erly compensate for the variation in sample extraction andionization eﬃciency of target molecules due to ion suppres-acyl-moiety at the sn-2 position of PC was amenable tostrategy to prepare IS compounds and probes for metabolic

sion/enhancement in LC-ESI-MS/MS analyses . These

incorporation into the phosphorylcholine fragment through52,53), producing a diagnostic
collision-induced dissociation

isotopically labeled analogs are also useful as probes tophosphorylcholine-d1 ion at m/z＝185, which facilitated thereveal the metabolic pathways of target molecules because,
as unaﬀected and metabolized forms, they are easily mea-

analysis of fatty acid metabolism in PC synthesis (Fig. 3B).

49)

Indeed, using synthesized tetradeuterated oxidized linoleic
acid (HODE-d4; hydroxyoctadecadienoic acid-d4) andsured separately from endogenous compounds according to
the m/z diﬀerence by LC-ESI-MS/MS, although it is still

HODE-d4-CoA,diﬃcult to prepare corresponding these labeled compounds.
To address these diﬃculties, several hydrogen-isotope

LPCAT2/LPLAT9, and AGPAT7/LPLAT10) were revealed
to be acyltransferases that directly transfer HODE-CoA into

exchange (HIE) methods have been reported by which one
can chemically synthesize deuterated fatty acids . VerylysoPC to produce PC species with a hydroxylated fatty51)acyl-moiety (Fig. 3C) . Given the validity of tetradeuter-recently, a novel HIE method to synthesize tetradeuterated

ated fatty acids, it would be possible to precisely dissect the

fatty acids was developed; the free fatty acid of interest is
derivatized into 8-amino-quinoline amides, followed by cat-aberrant lipid metabolism caused by ABCD1 disfunction in
X-ALD patients.

50)

alytic and K2CO3-mediated deuteration at the β - and α -car-

three

enzymes(LPCAT1/LPLAT8,

Conclusion

51)

bons of the fatty acid, respectively (Fig. 3A) . This HIE
method can be applied to a variety of fatty acids, including

To develop diagnostic and therapeutic methods for per-polyunsaturated fatty acids, cis/trans fatty acids, andoxisomal disorders, it is crucial to identify the causative

Fig. 3.

Synthesis and application of tetradeuterated fatty acid.

(A) Synthesis of tetradeuterated free fatty acid. Fatty acyl amides with 8-amino-quinoline (AQ) were deuterated at the β -carbonof the fatty acyl-moiety by heavy water (D2O) and palladium catalyst (Pd(OAc)2). Hydrogens at the α -carbon of the amides were
further exchanged with deuterium atoms from CH3OD by K2CO3, and tetradeuterated free fatty acids were obtained by hydrolysis.
(B) Production of deuterated phosphorylcholine in collision-induced dissociation (CID). In the process of CID, α -deuterium of
the tetradeuterated fatty acyl-moiety at the sn-2 position of PC was incorporated into the phosphorylcholine fragment, producing adiagnostic phosphorylcholine-d1 ion at m/z＝185.
(C) Identiﬁcation of acyltransferases that transfer hydroxyoctadecadienoic acid (HODE)-CoA into lysoPC. Three lysophospholipid
acyltransferases (LPCAT1/LPLAT8, LPCAT2/LPLAT9 and AGPAT7/LPLAT10) directly recognized a deuterated hydroxylated
linoleic fatty acyl-CoA (HODE-d4-CoA) as a substrate.

108

Medical Mass Spectrometry “Lipidomics” Vol. 6 No. 2

November 2022

molecules and/or metabolic machinery that explain the

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