November 2022

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Review

Investigating oxidized lipids in an omics way:
Oxidative lipidomics in biological applications using mass spectrometry
Zhen Chen , Xunzhi Wu , Hitoshi Chiba , Shu-Ping Hui *
1

1

2

1

1

Faculty of Health Sciences, Hokkaido University,
Kita-12, Nishi-5, Kita-ku, Sapporo, Hokkaido 060‒0812, Japan2
Department of Nutrition, Sapporo University of Health Sciences,
Nakanuma Nishi-4‒2‒1‒15, Higashi-ku, Sapporo, Hokkaido 007‒0894, Japan
Abstract

Oxidized lipids have been well known as potential markers of oxidative stress or other disorders in physiologi-

cal/pathological processes, such as cardiovascular diseases, neurodegenerative diseases, and even some cancers. Lipidomic
analysis is a modern and advanced approach to conducting informative elucidation for lipid molecules, which beneﬁts biomarker hunting, clinical determination, and biological pathway exploration. In recent years, as mass spectrometry technology advances, lipidomics is extending the investigating range towards oxidized lipids, which is called oxidative lipidomics.
This approach combines the complexity of the lipidome and focuses attention on oxidation. Because oxidized lipids areusually at low levels and artiﬁcial oxidation is undesired, oxidative lipidomics is more challenging than ordinary lipidomics, particularly in sample preparation, standards, and spectrum analysis. Nowadays, oxidative lipidomic analysis has been
applied to biomedical science for various diseases in clinics and animal experiments on disease models. This review aims
at the strategies and methods of oxidative lipidomics and the summary of current applications. The development of chromatographic-MS spectrometric technology is hopeful to allow a deeper and broader understanding of lipids and oxidation
concerning human health.
Key words: oxidative stress, lipid metabolism, lipid oxidation, hydroperoxide, LC-MS/MS

1. Introduction

age

2,3)

. Studies have shown that mammalian cells contain

Lipids are a large class of compounds that are easily sol-thousands of kinds of lipids. Moreover, with the continuous

uble in organic solvents and are heterogeneous in chemicaldevelopment of new technologies and methods, various

composition and structure, mainly including fatty acids andtheir naturally occurring derivatives as well as their biosyn-

new lipid molecules are still being discovered. According
to the comprehensive classiﬁcation system for lipids pro-1)

thetic and functionally related compounds . Lipids, known
as primary structural elements, represent a pretty varied

posed by the Lipid Metabolites And Pathways Strategy(LIPID MAPS), lipids are generally divided into eight cate-group of cellular components of biological membranes andare involved in intercellular signaling and energy stor-

gories: fatty acyls (FA), glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SP), sterol lipids (ST), pre4)

nol lipids (PR), saccharolipids (SL), and polyketides (PK) ,* Corresponding author

as shown in Fig. 1.

Shu-Ping Hui

The diversity of lipid structures endows lipids with a

Faculty of Health Sciences, Hokkaido University, Kita-12,
Nishi-5, Kita-ku, Sapporo, Hokkaido 060‒0812, Japanvariety of critical biological functions. Lipid metabolism
participates in regulating various life activities, such as pro-

Tel/Fax: +81‒11‒706‒3693
E-mail: keino@hs.hokudai.ac.jp
Received: April 10, 2022.

ducing energy, transporting materials, recognizing information, transmitting messages, being involved in cell develop-

Accepted: July 12, 2022.

ment, diﬀerentiation, apoptosis, and others. At the sametime, abnormal changes in lipid metabolism lead to inﬂu-

Epub August 8, 2022.
DOI: 10.24508/mms.2022.11.001
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Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Lipid classiﬁcation systems and the representative lipid molecules in each class. Fatty acyls (FA): 4Z,7Z,10Z,13Z,16Z,19Zdocosahexaenoic acid (DHA, C22:6n-3,6,9,12,15,18); glycerolipids (GL): 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycerol[DG(16:0/18:1(9Z)/0:0)]; glycerophospholipids (GP): 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine [PC(18:0/18:0)];sphingolipids (SP): N-(docosanoyl)-sphing-4-enine [Cer(d18:1/22:0)]; sterol lipids (ST): cholest-5-en-3 β -ol (cholesterol);prenol lipids (PR): 3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2E,4E,6E,8E-tetraenoic acid (retinoic acid); saccharolipids (SL): 2,3,4-O-triisobutyryl d-glucose (pennelliiside A); polyketides (PK): tautomycetin.

Fig. 1.

resulted in various oxidation products, such as hydroxides(‒OH), aldehydes (‒CHO), ketones (＝O), epoxides (–epo),

ences on cellular homeostasis, contributing to severe
illnesses covering from cardiovascular diseases (e.g.,coronary heart disease, hypertension, hyperlipidemia, arte5,6)

riosclerosis)

degraded small fragments, and others. These oxidation
products are known to cause biological membrane disrup-

to neurodegenerative diseases (e.g., Alzhei7,8), and even sometion, DNA damage, enzyme inactivation, and many other. During such dysregulated processes, speciﬁc

lipid mediators are generated, which can control diﬀerent

abnormal functions, and they have even been proposed to
be clinical biomarkers of some oxidative stress-related dis-

intracellular events.

eases . Therefore, it is of great importance to pay attention

mer s disease and Parkinson s disease)
9,10)

cancers13)

to such special lipid molecules.

Among such lipid mediators, one kind of molecule isdeﬁned as oxidized lipids. The unsaturated fatty chains are

Lipidomics, along with genomics, transcriptomics, pro-readily attacked by the reactive oxygen species (ROS) toform the oxygen-inserted structures, with which the lipid

teomics, and metabolomics, have been recognized as amolecules are classiﬁed as oxidized lipids. The oxidized
fatty chains can exist as free from, i.e., free fatty acids

many as hundreds or thousands) targets, which construct a
14)
global view of the status of the study objects . Techni-(FFA), or as esteriﬁed form, i.e., esteriﬁed fatty acids(EFA), such as glycerolipids, glycerophospholipids, or oth-cally, lipidomics is performed using liquid chromatography(LC) or gas chromatography (GC) coupled to mass spec-

ers. Lipid oxidation proceeds in vivo due to the endogenous
or exogenous free radicals. A key initial step of lipid oxida-

trometry (MS), nuclear magnetic resonance (NMR), or oth-holistic approach to simultaneously measuring multiple (as15)

tion is the generation of lipid hydroperoxides, which con-ers . From the acquired massive data covering structure
identiﬁcation to quantitative/semi-quantitative determina-

tains an added hydroperoxyl group and the C＝C doubletion, researchers can obtain comprehensive and systematic

11)

bond rearrangement, as revealed previously . According tocharacteristics of the lipids, understand their composition
and function, and reveal the interactions between the physi-

diﬀerent sources of oxidation, such as radical oxidation(e.g., thermal oxidation, auto-oxidation), enzymatic oxida-

ological/pathological changes and lipid metabolism. As lipi-tion (e.g., lipoxygenase), and singlet oxygen oxidation(e.g., photo-oxidation, inﬂammation), the formed hydroper-

domics represents the downstream products and tells theoxides (‒OOH) present varied and speciﬁc characteris-

applied in the whole organisms, organs or tissues, cells, andresearchers what indeed happened , it has been widely

12)tics , as shown in Fig. 2. The continued chain reactions

even organelles, to contribute to molecular physiology/
73

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Fig. 2.

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Structure variety of linoleic acid hydroperoxide isomers, namely 13‒9E,11E-HPODE, 9‒10E,12E-HPODE,
13‒9Z,11E-HPODE, 9‒10E,12Z-HPODE, 10‒8E,12Z-HPODE, and 12‒9Z,13E-HPODE.

pathology discovery, nutritional and environmental man-nance . Those methods include thiobarbituric acid reactive

agement, drug development, and other ﬁelds. In particular,
as one of the recently proposed strategies, oxidative lipid-

substances (TBARS) and malondialdehyde (MDA) test

omics focuses on the oxidized lipids and their oxidation/

gated diene test , and others. But the obtained results aredegradation products, connects lipids and oxidation, andusually considered as the indicator of the lipid oxidation

enhances the understanding of the role of oxidized lipids indegree, but not the detailed lipid changes.

16)

17‒21),, conju-

22,23)

ferrous oxidation in xylenol orange (FOX) test
24)

16)

cellular homeostasis and disease pathogenesis .

Besides, it is also needed to decide whether the targets

In the following sections, we will review the recent

are the total amount of certain lipid classes or individual

research progress in oxidative lipidomics in biological

species within single or multiple classes before analyzing.

applications, mainly including these parts: (1) the methods,
strategies, and workﬂow of conducting oxidative lipidom-

It is comparably easy to measure the total amount of certain

ics; (2) the applied samples and clinical diseases (or diseaseclasses, e.g., total aldehydes. While in terms of individual
species, previous separation and fractionation are com-models), and the uncovered ﬁndings associated with humanmonly needed, increasing labor work and increasing thehealth issues.

risk of artiﬁcial lipid oxidation. Moreover, more fractions
may lead to more complicated results.

2. Strategies and Methods of Conducting
Oxidative Lipidomics

For example, typically, after lipid extraction, the intact
lipids and the oxidized lipids are yielded as a mixture.

2.1. Traditional approaches to oxidized lipid analysis

Next, the mixture is puriﬁed predominantly on column

Because the lipid oxidation reactions are consecutive andchromatography with silica gel, alumina, or other sorbents.

the oxidized lipids are at a much lower level than the intact

Consequently, the unoxidized lipids with lower polarity are

lipids, in general, analysis of oxidized lipids is relatively

eluted using the nonpolar solvent, while the oxidized lipids
are collected with a stronger elution (with more polar sol-diﬃcult. Traditionally, analysis of oxidized lipids wasvents). One risk is that, for some highly polar (or even polymeric) oxidation products, the ﬁnal collection may be insuf-

based on indirect investigation of the lipid oxidation end
products, and those small molecules can be measured using
colorimetric assays, immunoassays, or electron-spin-reso-ﬁcient for removing them from the column, so the recovery
74

November 2022

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2of oxidized lipids is possible to be unsatisfactory.
Therefore, it is less diﬃcult to directly analyze the samples as the whole mixtures than to perform extra isolationand puriﬁcation for the oxidized lipid fractions from thecomplex substances, i.e., the sample matrix. Therefore,
methods with high selectivity, high sensitivity, and highspeciﬁcity are desired for achieving this aim.

2.2.

MS-based detection of the oxidized lipids

While other analytical methods require selection andadaption for measuring lipid oxidation products, MS technology overcomes the problems of sensitivity, speciﬁcity,
accuracy, and dynamic range to a great extent and enables
25)

high throughput analysis . Since lipid oxidation products
are always generated as a convoluted mixture with largely
varying levels and structural diversities, MS-based detection of the oxidized lipids is considered to be a promising26)

approach . While the combination of chromatography andMS have been applied in biology and clinical samples as
essential platforms in analyzing oxidized lipids, such as
GC/MS, LC/MS, or even supercritical ﬂuid chromatography (SFC)/MS, and nowadays, LC/MS technique (particularly LC coupled to tandem MS, i.e., LC/MS/MS) is thedominant approach for oxidative lipidomic applications.
Structurally, oxidized lipids refer to the extra oxygen atoms
attached to the lipid molecules, including the insertion of

Scheme 1.

the peroxyl/hydroperoxyl/epoxyl group and the deletion of27)

Workﬂow of oxidative lipidomics in biologicalapplications.

the C＝C double bond . Therefore, using MS (especiallyhigh-resolution MS), the lipid molecules with oxidized

to the non-targeted analysis.

functional groups can be captured, and using MS/MS, thestructure conﬁrmation can be conducted, including the con-

2.3.

ﬁrmation of oxygen existence, the determination of oxidiz-

Workﬂow of MS-based oxidative lipidomics

As the same as ordinary lipidomics, oxidative lipidomics

ing position, and the discovery of speciﬁc fragmentation(typically using LC/MS) also follows the steps below

from precursor ions to product ions.

(Scheme 1). Sampling: collection of clinical, animal, plant,

In general, there are two strategies to analyze oxidized lip28)
ids: the targeted analysis and the non-targeted analysis . For

cell, or other samples, as well as necessary and appropriate
pretreatments; Extraction: commonly being accorded toBligh-Dyer, Folch, Matyash, or others, with monophase,the known oxidized lipids (especially when they are also29‒32); LC/MS analysis: injecting the carefully prepared lipid samples into the instru-

interesting to investigate), the targeted strategy is usuallybiphase, or triphase extraction

conducted, such as the oxidized free fatty acids (oxFFA),
esters (oxCE), and oxidized phospholipids (oxPL) . Whilements using optimized conditions, which can be either targeted or non-targeted according to experiment design; Raw

for simultaneously viewing every possibly detectable lipid
species within multiple classes , the non-targeted strategy isdata analysis & Information integration: interpreting thedata of oxidized lipids, digging the deep meaning, and con-preferable. Most of the studies on oxidized fatty acids andnecting to the aim(s) of the research. It should be paidoxidized carnitines are conducted by targeted analysis, while

extreme attention to that, compared with ordinary lipids,

oxidative lipidomic studies focusing on more classes belong

the oxidized lipids, usually at a very low level, are moreoxidized cholesterol or other oxysterols, oxidized cholesteryl
26)

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Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Fig. 3.

Characterized MS signals of CL(18:2)4-OOH compared with CL(18:2)4.

unstable. Therefore, from sampling, all the operations
should avoid auto-oxidation and degradation. After collec-

samples is needed using solid-phase extraction (SPE),

tion from the living organism, the samples should bestudies focusing on the small molecules as the oxidation/

extracted immediately or be quickly frozen using dry ice ordegradation products of lipids, as those compounds are not
as non-volatile as lipids, long-time evaporation at high tem-

which should also be carefully operated. Besides, for theliquid nitrogen and kept in an atmosphere of noble gas inglass containers; homogenization and vortex with ice-coldperature and low pressure may cause the target loss.

solvents at the lowest temperature whenever practicable;

With appropriate sampling and pretreatment, the injected
samples were subjected to MS detection, in which the oxi-addition of antioxidants for protection, typically butylatedhydroxytoluene (BHT). Not only air/oxygen but also proteins should be noticed: on the one hand, the hydroperoxide

dized lipids are theoretically ionized and fragmented. The
resulted MS and MS/MS signals provided structural infor-

groups of the oxidized lipids can react with certain proteins
to form covalent bonds, causing false-negative results; onmation for identiﬁcation. Taking CL(18:2)4-OOH as an
example (Fig. 3), in our experiment, HR-MS showed thatthe other hand, the oxygenated fatty acids can be artefactu-

the [M−H] of oxidized CL(18:2)4 (m/z 1479.9526) was−

33)

ally produced by lipoxygenases , therefore causing
false-positive results. Sometimes the puriﬁcation of the

31.9914 Da more than the intact CL(18:2)4 (m/z1447.9612),
76

suggestingtheelemental

composition

November 2022

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2−

C81H141O19P2 , which can be assigned as CL(18:2)4＋2[O].

stress and appeared in a dysregulated state in diﬀerent dis-

The further MS spectrum of the oxidized CL(18:2)4 gives−
not only m/z 695 and m/z 831 (assigned as [PA36:4‒H]

eases, researchers express an increasing interest in theseand [M‒PA36:4‒H] , respectively), but also m/z 727 and−−m/z 863 as the [PA36:4‒H＋2O] and [M‒PA36:4‒H＋2O] ,for certain diseases. Based on lipid class, oxidized struc-respectively. Moreover, m/z 1391 suggested the position ofhydroperoxidation at the C-13 position, which was a char-the oxidized lipids into three classes: (1) lipids containinghydroxy fatty acids, including 2-hydroxy-ceramides,

acteristic fragmentation pattern of hydroperoxide, as
34,35). Therefore, by HR-MS and MS/
reported previously

2-hydroxy-sphingomyelins, 2-hydroxy-cerebrosides, 2-hydroxy-gangliosides, and hydroxized acyl-L-carnitines; (2) lipids

MS, we can clarify the structure of the oxidized lipid

containing PUFA peroxidation products, including oxidized
phosphatidylcholine (ox-PC), oxidized phosphatidyletha-

2oxidized lipids and consider them potential biomarkers−

28)ture, and formation routine, a recent review

molecular species, which is critical for understanding thecharacteristics and building the in-house library. Neverthe-

divided allnolamine (ox-PE), oxidized phosphatidylserine (ox-PS),
oxidized cardiolipin (ox-CL), and ox-CE; (3) oxidized freeless, a better way to elucidate the structure of the oxidized
lipids, especially distinguishing hydroperoxy, (di)hydroxy,and (hydroxy)epoxy compounds, is to compare their frag-cholesterol (oxysterols). In this review, we would like toments using authentic standards. But in most cases, unfortunately, such lipid standards are not available. Therefore, itapproach, focusing on the ﬁndings concerning diseases,
aging, injuries, and other factors. A collection of representa-

is of great importance to set up the fragmentation databasetive works related to oxidative lipidomics in biologicaland to recognize the MS/MS pattern.

(clinical, animal, or cell levels) samples is listed in Table 1.

Such identiﬁcation and the subsequent quantitation (orsemi-quantitation) can be executed manually or using auto-

3.1.

summarize the applications of the oxidative lipidomic

Oxidative stress-related metabolic diseases

Oxidative stress is one of the main factors involved in themated software. Besides the workstation software provided
by the manufacturers (e.g., Xcalibur by Thermoﬁsher Sci-

pathogenesis of cardiovascular disease (CVD), which is theentiﬁc, Inc.), there are increasing data process platforms toleading cause of morbidity and mortality in the world .

conduct chromatographic peak alignment, deconvolution,

The overproduced ROS not only breaks the balance

peak identiﬁcation from the commercial solutions (e.g.,
LipidSearch, Progenesis QI) to the open-source programs

between oxidative stress and antioxidant defense but also
directly attacks the intact lipid molecules to generate oxida-(MZmine, LipidFinder). Typically, the open-source software MS-DIAL developed by RIKEN enables the data-in-

tive lipid species.

37)

Alcoholic and non-alcoholic fatty liver diseases
Both alcoholic fatty liver disease (AFLD) and non-alco-

3.1.1.

dependent MS/MS deconvolution for comprehensive lipid36)

omic analysis . It should be noted that the users usuallywish to build their own in-house libraries to get the optimal

holic fatty liver disease (NAFLD) mean the accumulationof fat in the liver, resulting in an enlarged and damaged
liver and hepatic steatosis. Unlike AFLD, which is initial-

matches of spectrum comparison. Finally, the obtained
information on oxidative lipids is required to be integrated
with a biological system or disease mechanism, just as theized by drinking a large amount of alcohol, NAFLD iscaused by a build-up of fat. Currently, NAFLD and non-al-intact lipidomics.

coholic steatohepatitis (NASH) have become common
38)

causes of chronic liver disease in the world , which have

3. The Applied Samples and Clinical Diseases(or Disease Models)39)

been reported to be associated with oxidative stress . In
terms of lipid oxidation, in both human and mouse models
with either AFLD or NAFLD, researchers have found oxi-

Lipids have been reported to be involved in a variety ofphysiological and pathological procedures. As the analyti-

dized lipid species, which mainly include the addition of

cal techniques develop, this ﬁeld is rapidly growing, andcarbonyl, hydroxyl, and hydroperoxyl groups to TG, PC,

researchers are expanding the coverage of the lipidome

and FFA

from the intact lipids to the oxidized lipids. Since there

3.1.2.

40‒43).

Diabetes mellitus

have been many reports on the observation of oxygenatedDiabetes mellitus includes type 1 diabetes (accounted forlipid molecules, which were inherently linked to oxidative

approximately 10%) and type 2 diabetes (accounted for77

November 2022

Table 1.
Entry

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Diseases (and disease models) investigated using the lipidomic approach for oxidized lipid species

Disease/disorders

Biological sample
OriginAnalytical method

Specimen

Strategy

Extraction protocol

Platform

Oxidized lipids investigated

Ref.

1

Alcoholic fatty liver disease

Mouse

Liver

Non-targeted

Bligh-Dyer

LC/MS

TG＝O

40)

2

Alcoholic liver disease

Human

PlasmaTargeted

N/A

LC/MS

PC-OH, PC-OOH

41)

3

Non-alcoholic steatohepatitis

Mouse

Liver, kidney

Non-targeted

Folch

LC/MS

TG-OOH, PC-OOH

42)

4

Nonalcoholic fatty liver disease

Human

Liver

Targeted

isopropanol/hexane

LC/MS

FFA-OH, FFA-OOH

43)

5Type 2 diabetes mellitus

Mouse

Serum, liver, brain, ventricle, atrium, renal medulla,
renal cortex, spleenNon-targeted

Folch, Bligh‒Dyer

LC/MS

TG-OOH, PC-OOH,
PE-OOH, PI-OOH

46)

6Type 2 diabetes mellitus

Zebraﬁsh

PlasmaNon-targeted

Bligh‒Dyer

LC/MS

PC-OOH, PE-OOH,
PC-CHO47)

7

Type 2 diabetes mellitus

Mouse

Plasma, urine

Targeted

MethanolLC/MS

Carnitine-OH

48)

8

Type 1 diabetes mellitus

Mouse

PlasmaTargeted

Methanol, with83)
derivatization

LC/MS

Carnitine-OH

49)

9

Obesity

Mouse

Liver

Targeted

Methanol

SFC/MS

PC-OH, PC-OOH,
PC-epo50)

10Obesity

Mouse

Liver, subcutaneous
adipose tissue

Targeted

Folch

LC/MS

Cholesterol-OH, cholesterol＝O, cholesterol-epo

51)

11

Metabolic syndrome (geneticallyobese spontaneously hypertensive)

Rat

PlasmaTargeted

Methanol, with SPE

LC/MS

FFA-OH, FFA-OOH

54)

12

Chronic kidney disease

Human

PlasmaTargeted

MatyashLC/MS

PC-CHO, PC-COOH

55)

74)

43)

13

Immunoglobulin A nephropathy

Human

Serum, urine

Targeted

SPE

LC/MS

FFA-OH, FFA-OOH,
FFA-epo56)

14

Alzheimer s disease

Human

Serum

Targeted

MatyashLC/MS

PC-CHO

58)

15

Alzheimer s disease

Human

Brain

Non-targeted

Ethanol phosphate
buﬀer

MS

SM-OH

59)

16

Parkinson s disease

Rat

Substantia nigra, plasma

Non-targeted

Folch, Bligh‒Dyer

LC/MS

EFA-OH, EFA-epo,
oxCL

61)

17

Parkinson s disease

Human

PlasmaNon-targeted

Methanol, withhydrolyzation and84)
derivatization

GC/MS

FA-OH, cholesterol-OH,
cholesterol＝O,

62)

18

Coronary artery disease

Human

PlasmaNon-targeted

Modiﬁed Folch

85)

LC/MS

OxPA, oxPC, oxPE

63)

19

Coronary artery disease

Human

PlateletNon-targeted

Methanol

LC/MS

PC-CHO, PC-COOH

64)

20

Acute coronary syndrome

Human

PlasmaTargeted

Folch

LC/MS

PC-CHO, PC-COOH

65)

21

Atherosclerosis

Human

Plasma, atherosclerotic
plaquesTargeted

Folch

LC/MS

OxPC

66)

22

Peripheral atherosclerotic
disease

Human

Arterial intima tissue

Targeted

Bligh-Dyer,
86)with SPE andderivatization

GC/MS

cholesterol-OH

67)

23

Kawasaki disease

Human

Serum

Targeted,
non-targeted

N/A

LC/MS

PC-OH, PE-OH, PI-OH

68)

24

Non-pancreatic neuroendocrine
tumor

Human

PlasmaNon-targeted

Methanol/ethanol

LC/MS

FFA-OH, LPC-OH

70)

Hepatocellular carcinoma

Human

Liver

Targeted

Modiﬁed Bligh-Dyer

LC/MS

CL-OH

71)

N/A

MS
imaging

CL-OH, CL-OOH

72)

26

Oncocytic thyroid tumor

Human

Thyroid

Non-targeted27

Medium-chain acyl-CoA dehydrogenase deﬁciency

Human

Blood

Non-targeted

Methanol

LC/MS

PC-COOH

73)

28

Septic inﬂammatory

Mouse

Bronchiolar alveolar
lavage ﬂuid, serum

Targeted

N/A

LC/MS

FFA-OH, FFA-epo

74)

29

Periodontal disease

Human

Saliva, serum

Targeted

Hexane/methyl tbutyl ether

LC/MS

FFA-OH75)

30

Aging and ischemia/reperfusion

Mouse

Heart

Non-targeted

Folch

LC/MS

OxCL

77)

31

Transient hepatic ischemia

Rat

Liver

Targeted

Folch

LC/MS

OxCL

78)

Lung

Non-targeted

Folch

LC/MS

CL-OH, PS-OH

79)

LC/MS

FA-OH, FA-OOH

80)

LC/MS

PC-CHO, PC-COOH,
PC＝O, CE-OH

81)

32

Hyperoxia

Mouse

33

Cardiac surgery

Human

PlasmaTargeted

Methanol, with hydrolyzation and SPE

34

Iatrogenic plaque disruption

Human

Blood

Targeted

Folch

N/A: Not available (not mentioned in the literature).

78

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Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

44)Parkinson s disease (PD), oxidative stress also plays a keyrole in the degeneration of dopaminergic neurons and dis-

approximately 90%) . Both have been recognized to berelated to oxidative stress, especially for diabetic complications . Animal studies on diﬀerent samples (blood, urine,and multiple organs and tissues) have proved the accumula-ruption of the physiologic maintenance . Using non-tar-tion of TG-OOH, PC-OOH, PE-OOH, and PI-OOH species

expressed the oxidized lipid molecules covering oxFA,
61,62), as well as their potential as valuoxCL, and oxysterols

45)

60)

geted approaches via LC/MS or GC/MS, studies haveand the elevation of carniby non-targeted approach
48,49). It is noted that the lipid
tine-OH by targeted approachable biomarkers.

hydroperoxides might be the potential biomarkers for not

3.1.6.

only diabetes itself but also diabetic complications.

eases

46,47)

3.1.3.

Other metabolism disorder-related vascular dis-

Besides the reviewed conditions above, oxidized lipid-

Obesity

For obesity, mouse model experiments revealed a series
50)of PC oxidation products and various oxidized choles-

omics has also been applied to other metabolic disorders. In
63,64)

clinical, coronary artery disease (CAD)

terol molecules (oxysterols), such as cholesterol-OH, cho51)
lesterol＝O, and cholesterol-epo . Although oxysterols had

65)

66)

syndrome (ACS) , and atherosclerosis, acute coronary

have been proved

to be connected to oxidized phospholipids. Especially, thePC oxidation products PC-CHO and PC-COOH species

been studied as the potential biomarkers of rare genetic dis52)
eases, such as Niemann-Pick disease type C and cerebro-

have been proposed by both non-targeted and targeted53)

tendinous xanthomatosis , they have also been recognizedmethods in CAD patients. Moreover, one study measured
cholesterol-OH species using GC/MS in the arterial intima

as the indicators for the excess and oxidation of cholesterol.
Additionally, a targeted lipidomic analysis of rat plasma ofa metabolic syndrome model suggested FFA-OH and FFA-

tissue from peripheral atherosclerotic disease (PAD)
patients, and found that these oxysterols were issue andplasma were highly related to systemic inﬂammatory activ-

OOH species as feasible biomarkers for diagnostic and
54)

67)

therapeutic/pharmacological evaluation .

ity . In addition, a blood vessel inﬂammation syndrome,

3.1.4.

As one of the most severe renal diseases, chronic kidney

Kawasaki disease (KD), has also been reported to link to
68)
oxidized phospholipids . The untargeted lipidomic proﬁl-disease (CKD) involves a gradual loss of kidney function,

ing and targeted analysis indicated that oxPC species werewhich is also called chronic kidney failure. By using a

related to coronary arteritis, suggesting that the oxidized

quantitative lipidomic method, a study found that the CKD

phospholipids activated the inﬂammatory signals in KD.

patient plasma samples showed an altered oxPC signature
discrimination between the conditions with or without peri-

3.2.

Renal diseases

55)

odontal comorbidity . Another study on immunoglobulin
A nephropathy (IgAN) measured the oxFFA proﬁle (includ-

between oxidation and reduction happened due to theover-production of active oxygen by tumor cells and theing FFA-OH, FFA-OOH, FFA-epo species) in human serumand urine samples using a targeted analysis with SPE pre-69)
abnormal oxidation-reduction control , oxidative lipidom-

treatment . The authors also proposed these oxFFA spe56)

ics has been applied to some cancer studies in the clinic,
70)
namely, non-pancreatic neuroendocrine tumor , hepatocel-

cies as biomarkers for IgAN diagnosis and evaluation index
of n-3 fatty acid supplementation.
3.1.5.

Cancers

Since researchers have realized that the unbalance

71)
72)
lular carcinoma , and oncocytic thyroid tumor . In addition to FFA-OH and LPC-OH, the oxCL species (e.g.,

Neurodegenerative diseases

In the past decade, oxidative stress has been considered
as a major role in the process of neurodegenerative dis-

CL-OH and CL-OOH) were found as elevated indexes, andeases, for example, Alzheimer s disease (AD) . The accel-

mitochondrial dysfunction in tumor cells.

such abnormality might be explained as the uncontrolled

57)

erated oxidation of glycated proteins might contribute to

3.3.

the accumulation of advanced glycation endproducts inamyloid plaques. In terms of oxidative lipidome, the oxi-

Other diseases

Oxidative lipidomic analysis has also been applied to73)
medium-chain acyl-CoA dehydrogenase deﬁciency , sep-

dized phospholipids have been revealed, such as PC-CHO
58)
species in human serum and SM-OH species in human

74)

75)

tic inﬂammatory , periodontal disease , and others. A
representative and well-developed method is the triple

59)

brain . For another progressive nervous system disorder,
79

November 2022

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

quadrupole MS-based quantitative determination of
FFA-OH, FFA-OOH, and FFA-epo as the targeted approach,dards but also the library establishment, even including thein-silico calculation, prediction, and simulation. Further-which has been used for blood (either plasma or serum) andmore, the expansion of bioinformatics will contribute to aother body ﬂuids (e.g., urine and saliva).

better understanding of lipid dynamics and integration ofother omics (e.g., genomics and proteomics). Another

Surgery- and injury-induced disorders

3.4.

aspect is the development of imaging, which can be moreintuitive and visually direct for speciﬁc studies and innova-

Not only diseases but also surgery- and injury-induced

disorders are practicable for oxidative lipidomic analysis.

tive diagnoses.

Ischemia/reperfusion (I/R) is a typical injury that causes

5. Summary

76)

oxidation damage to organs . In animal I/R organ samples,both non-targeted and targeted studies discovered oxCL
77,78)

species as signiﬁcantly increased indicators

As a branch of lipidomics, oxidative lipidomics focuses. The

on the oxidized lipid molecules of diﬀerent lipid classes,
including lipid-OH, lipid-OOH, lipid-epo, lipid＝O, andhydroxyl CL molecules were also found in the lung of thehyperoxia mouse model, as well as hydroxyl PS (PS-even the degraded lipid oxidation products and other79)OH) . From human plasma of cardiac surgery, targeted
lipidomics determined FA-OH and FA-OOH as damage

related molecules. These characterized lipid species havebeen recognized as useful biomarkers for both diagnosis
and treatment evaluation. The application of oxidative lipi-

markers, but since the pretreatment included hydrolyzation,
the source of these oxidized fatty acyls remained

domics by chromatographic separation and MS detection
covers both oxidative stress-related metabolic diseases and80)

unknown . Moreover, in the patient blood of iatrogenic
plaque disruption, a series of oxPC, including PC-CHO,

other disorders, from animal experiments to clinical tests.

PC-COOH, and PC＝O, were quantitatively investigatedCurrently, compared with ordinary lipidomics, i.e., intact

together with CE-OH .

lipidomics, oxidative lipidomics faces more challenges,

81)

especially the lack of standards and compound database.

4. Limitations and Prospects of Oxidative
Lipidomics

Nevertheless, it is a promising way to explore the ﬁeld ofbiomedical studies. As MS technological advances, a

Although studies on oxidative lipidomics are increasing
rapidly, some limitations are worth noticing. Basically, oxi-

deeper and broader understanding of lipids and oxidation
concerning human health will be achieved.

dative lipidomics shares all the boundaries of ordinary lipi-

Acknowledgments

domics. Like metabolomics and other downstream omics,

This work is supported by JSPS KAKENHI Grant Num-

oxidative lipidomics lacks the interpretation of upstream
information and underlying mechanism (metabolic path-bers 18K07434, 19K07861, and 21K14799.

ways). In terms of operation, oxidative lipidomics always

Conﬂict of Interest

82)

faces the challenges of sampling, pretreating, and storing ;All authors declare that they have no conﬂict of interest.

it is also hard to deal with the distinguishment of artiﬁcial
oxidation products. For data processing, there are usuallydiﬃculties in accomplishing structural validation and con-

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