June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Review

Oxidized lipid species in lipoproteins:
Signiﬁcance and analytical considerations
Rojeet Shrestha , Hitoshi Chiba , Shu-Ping Hui *1

1

Abstract

2

1

Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo, Hokkaido 060‒0812, Japan2
Department of Nutrition, Sapporo University of Health Sciences,
Nakanuma Nishi-4‒2‒1‒15, Higashi-ku, Sapporo, Hokkkaido 007‒0894, Japan
Lipids are ubiquitous in the human body, playing diverse biochemical and physiological functions. Unfortu-

nately, lipids are susceptible to oxidation in vivo by free radicals. Lipid hydroperoxides (LOOH) are one of the primary
products of lipid peroxidation which play a crucial role in the pathogenesis of oxidative stress, inﬂammation, and atherosclerosis. Understanding the underlying molecular mechanism of these diseases is critically important for risk stratiﬁcation,
prevention, and therapeutic intervention. Despite its importance in the atherosclerotic process, the qualitative and quantitative evaluations of LOOH in the biological samples have been largely limited, because of structural variability, instability
and rapid clearance from circulation. Application of liquid chromatography/mass spectrometry (LC/MS) has revolutionized
the study of lipidomic and eased the identiﬁcation and quantiﬁcation of hundreds of molecular species of oxidized lipids
derived from fatty acids, cholesteryl esters, triacylglycerol and phospholipids in various biological samples. In this review,
we will be discussing oxidized lipid species in human lipoproteins, its analytical consideration and signiﬁcance. An overview of lipids and its oxidative products in lipoproteins, technological advances its identiﬁcation and quantiﬁcation usingmass spectrometry, and its possible linkage to disorders of lipid metabolism will be discussed.
Key words: LC/MS, cholesteryl ester, triacylglycerol, hydroperoxide, phospholipid, oxHDL, oxLDL

Introduction

logical need. Similarly, lipids particularly phospholipids

Lipids are ubiquitous in the human body, playing diverse(PL) are essential components of biological membranes.

biochemical and physiological functions. Most importantly,

The membrane also contains cholesterol that modulates theﬂuidity of membrane. It also serves as a precursor for sev-

they serve as a reserve source of energy. Excess calories inour body are converted into fatty acids (FA), which subse-

eral steroid hormones, vitamin D and bile salts. As beinghydrophobic in nature, except for free FA, which are trans-

quently are esteriﬁed with glycerol forming triacylglycerols(TG) and stored particularly in adipose tissue. When there

ported binding to albumin, lipid molecules are transported
in systemic circulation in the form of lipoproteins. Unfortu-

is a demand for energy, TG is mobilized by cellularenzymes into FA, which are then distributed via blood to fat
utilizing tissues like muscles and heart to fulﬁll the physio-

nately, lipids are equally associated with several diseases.
Any distortion or alternation in this metabolic pathway canlead to adverse conditions and some of them remain as a
global health problem in today s clinical practice. In addi-*Corresponding author
Shu-Ping Huition, unsaturated hydrocarbons of lipids are susceptible toFaculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo, Hokkaido 060‒0812, Japan

oxidation by reactive oxygen species (ROS) generated byin our body. There is increasing evidence that such oxidized

Tel & Fax: +81‒11‒706‒3693
E-mail: keino@hs.hokudai.ac.jp

lipids play a crucial role in the pathogenesis of oxidative

Received: January 14, 2020. Accepted: March 11, 2020.

stress, inﬂammation, atherosclerosis, and coronary heart

Epub April 15, 2020.

disease (CHD). Understanding the underlying molecular

DOI: 10.24508/mms.2020.06.003

mechanism of these diseases is critically important for risk
2June 2020

Medical Mass Spectrometry Vol. 4 No. 1

stratiﬁcation, prevention, and therapeutic intervention. In

LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C)

this review, we will be discussing oxidized lipid species inhuman lipoproteins, its analytical consideration and signiﬁ-

are routinely measured for the diagnosis and monitoring ofvarious dyslipidemia and also to evaluate the risk of athero-cance. An overview of lipids and its oxidative products insclerotic cardiovascular disease (ASCVD). There is almost

lipoproteins, technological advances its identiﬁcation andquantiﬁcation using mass spectrometry (MS), and its possi-

universal agreement that LDL plays a central role in theble linkage to disorders of lipid metabolism will be dis-pathogenesis of atherosclerosis and coronary artery disease(CAD). The overall relationship between LDL-C andcussed in brief.

ASCVD is curvilinear. Therefore, LDL-C is considered as a

Lipids and Lipoproteinsprime target of therapy for the primary prevention of
5)
CAD . HDL-C, on the other hand, protects against the

Exogenous or dietary fats (mainly TG) are carried into
systemic circulation by chylomicrons, whereas endoge-

development of atherosclerosis and low levels of HDL-C
are associated with an increased incidence of ASCVD in

nous, that is fat synthesized de novo by the liver, are carriedby very low-density lipoproteins (VLDL). As these TG-rich

epidemiological studies . Due to technical limitations andcontroversy in the measurement of LDL-C, calculation of

lipoproteins (TRL) passes through circulation, Apo CII acti-

non-HDL-cholesterol (nonHDL-C), which includes choles-

vates the lipoprotein lipase, which is an extracellular

terol in LDL and other proatherogenic TRL, is gaining its

enzyme anchored by heparan sulfate to the capillary wall.

popularity in clinical practice. Individuals with abdominal

This enzyme rapidly hydrolyzes TG in TRL into free FA

obesity, metabolic syndrome, or diabetic lipid disorders
often have increased TG, low HDL-C, and non-increased

6)which is then taken up by the adipose tissue for storage,
and by muscles for its utilization . As TG get utilized, thecalculated LDL-C. These patients produce highly athero-

VLDL becomes much smaller and dense. Further, TG is

genic lipoproteins such as VLDL and IDL as well as small
dense LDL (sdLDL) particles. For this reason, non-HDL-C

1)transferred to HDL in exchange for cholesteryl ester (CE).
This process is mediated by cholesteryl ester transfer pro-is a better marker of cardiovascular disease (CVD) in bothtein (CETP). Some amount of these remnant VLDL, also
called as intermediate-density lipoprotein (IDL), are taken

primary and secondary prevention studies. Therefore, the2018 ACC/AHA guideline endorsed its routine use alterna-

up by the liver via ApoE receptor-mediated endocytosis. In

tive to LDL-C .
7)

We have been known for several decades that almost half

the remaining, TG are further utilized by hepatic lipase
forming cholesterol-rich low-density lipoprotein (LDL),of all myocardial infarction (MI) occurs among individuals

which functions to deliver cholesteryl ester (CE) to peripheral tissue. The LDL are endocytosed by extra-hepatic tis-

who have average if not low level of plasma cholesterol.

sue via ApoB receptor-mediated endocytosis . The synthe-

to reveal biomarkers that can provide information about

sis of the LDL receptor must be under negative feedback

future cardiovascular risk above and beyond cholesterol .

regulation of intracellular cholesterol concentration in orderto prevent excessive deposition of CE in peripheral tis-

Measurement of oxidized lipoproteins is of great interest tosues . High-density lipoproteins (HDL), on the other hand,
are responsible for reverse-cholesterol transportation. Thattarget.

Therefore, a considerable amount of research is being done

2)8)

improve the risk stratiﬁcation and possibly as a therapeutic3)

is, it receives the cholesterol from the peripheral tissues by
the process mediated by ATP-binding cassette transport pro-

Oxidized Lipoproteins

tein (ABCA1). The cholesterols are then rapidly esteriﬁed
to CE using FA from Sn-2 position of phosphatidylcholine. Therefore, oxidized lipoproteins
have received great attention, both to understand the patho-(PC) catalyzed by lecithin cholesteryl ester transport pro-physiology of atherosclerosis and as a potential biomarker

Oxidation of lipoproteins is another key factor that can
9,10)elicit the atherogenesis

4)

teins (LCAT) . HDL thus carries excessive cholesterol

for risk stratiﬁcation of ASCVD. The original idea of

from the peripheral tissues back to the liver for its excretion

proatherogenicity of structural modiﬁcation of circulating

and conversion to bile salt. An overview of lipoprotein

LDL was reported by Brown and Goldstein . Various ROS

metabolism is shown in Fig. 1.

generated during the normal cellular metabolism or induced

11)

by various environmental factors like pollutants, smoking,In clinical practice, serum TG, total cholesterol (TC),3

June 2020

Fig. 1.

Medical Mass Spectrometry Vol. 4 No. 1

Overview of lipoprotein metabolism.

ApoB-R: ApoB100-receptor, ApoE-R: apo E-receptor, ATGL: adipose tissue triglyceride lipase, CE: cholesteryl ester,
CETP: cholesteryl ester transfer protein, ChREBP: carbohydrate-response element-binding protein, CM: chylomicrons,
FA: fatty acids, HL: hepatic lipase, LCAT: lecithin cholesterol acyltransferase, LD: lipid droplets, LPL: lipoprotein lipase,
MTTP: microsomal triglyceride transfer proteins, oxLP: oxidized lipoproteins, R-CM: Remnant chylomicrons, SREBP:
sterol regulatory element-binding proteins, TG: triacylglycerol.
12)

toxin, and drugs can oxidatively modify the lipoproteins.

the process of atherogenesis . Finally, these foam cells

The oxidized LDL (OxLDL) may trigger the inﬂammation

accumulate, releasing various growth factors and cytokines

in the local arteries. In response to endothelium injury, thethat stimulate the proliferation of smooth muscle cells andaccumulate more lipids and ultimately activate the throm-

monocytes adhere to endothelial cells, and then move to thesub-endothelium layer and ﬁnally get transformed into mac-

13‒18)

botic event resulting in atherosclerosis

rophages. The macrophages contain non-speciﬁc, low-aﬃn-. It is now wellevident that LDL oxidation can occur in vivo and OxLDL
have been identiﬁed in human atherosclerotic lesions, fur-

ity scavenger receptors mainly CD36 that can bind with

19,20)

ther supporting the involvement of OxLDL in ASCVDOxLDL. Unfortunately, these scavenger receptors are notregulated by the cellular cholesterol level, therefore, macro-.

phages consume excess amounts of modiﬁed lipoproteins

Thus, it appears that the measurement of OxLDL could
serve as a better marker for atherosclerosis. Direct detec-

and ultimately becoming foam cells, which is a hallmark intion of OxLDL can be done with anion-exchange chroma4

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

tography, size-exclusion chromatography, agarose gel andcapillary electrophoresis and has been reviewed by Yama-

enzymatic or non-enzymatic reactions. Many oxidized

guchi et al . Considerable numbers of enzyme immunoassay (EIA) speciﬁc for various epitopes of OxLDL are cur-

FA have been documented in vitro and many of them are

rentlywell documented in human atherosclerotic lesions

forms of cholesterol and its ester, TG, PL, glycolipids, and21)

also identiﬁed in vivo. Presence of oxidized lipids has been
39,40)

available.

Antibodies
directedagainstmalondialdehyde (MDA)-LDL or OxPL-LDL are widely.

used in the research settings. Analytical importance andLipoproteins are predominantly composed of PL, CE, and
TG. Apart from the structural modiﬁcation apoproteins, oxi-

limitation of various assays for the direct evaluation ofdized lipoproteins can contain oxidized PL, CE and TG

OxLDL have been appropriately reviewed by Itabe and[Fig. 2]. Since the presence of oxidized lipid species can

22)

Ueda . Although accumulating information supports theimportance of OxLDL measurement as a potential bio-

increase its atherogenicity, studies on these oxidized lipids
may provide valuable information on the process of athero-marker for the risk stratiﬁcation of ASCVD, the clinical rel-genesis. For instance, CE hydroperoxides (CEOOH) are theevance of measurement of OxLDL is yet to be established;major biologically active components of OxLDL, which inturn stimulate a wide variety of cellular and molecular pro-hence, are not in routine use. Alternatively, EIA to detectautoantibodies against OxLDL are available which cor-41)

cesses involved in atherosclerosis . Oxidized lipids are
believed to play an important role in inﬂammation, oxida-

relates with the direct OxLDL measurement. However, alarge prospective clinical study on the Framingham Oﬀ-

42‒48)

tive stress, immune response and tumorigenesis

spring Study failed to show its association with the inci-.

The precise mechanism of the lipid peroxidation (LPO)
in vivo is largely unknown. FA, particularly polyunsatu-

23)

dence of CHD or CVD . Measurement of OxLDL andanti-oxLDL antibodies for the risk stratiﬁcation of ASCVD

rated fatty acids (PUFA), are vulnerable for the peroxida-24)

and its future implication has been recently reviewed .
MS-based approaches for the analysis of oxidative modiﬁ-tion . It is generally believed that ROS generated in our
body, either during normal metabolism or due to environ-49)

cation of apoproteins will not be discussed here but has

mental factors, are responsible for non-enzymatic oxidative

25)

50)

been excellently reviewed by Afonso et al .

modiﬁcation of biomolecules . In addition, oxidative

Although OxLDL are primarily blamed for atherosclero-stress induced by waning antioxidant systems may play asis, increasing evidence is revealing the potentiality of

crucial role in LPO. The oxidation is usually initiated by

VLDL and IDL to initiate the process of atherosclerosis;

the abstraction of a hydrogen atom from a carbon adjacent

therefore, role of the oxidized form of these TRL cannot be

to double bonds, resulting in the formation of conjugated26‒29). Both VLDL and IDL have been isolated from30‒32). Further-dienes. Subsequently, oxygen molecule is incorporated into

the human aorta and atherosclerotic plaquesit, leading to the formation of LOOH. Therefore, LOOH is

more, oxidized TRL are considered as proatherogenic andconsidered as an early product indicator of LPO . LOOH
can further degrade to the ﬁnal products like malondialde-

negated51)

can increase cellular CE uptake by macrophages resulting
in the formation of foam cells . On the other hand, a study

33)

hyde (MDA) and 4-hydroxynonenal. Furthermore, LOOH

by Bowry et al. indicated that HDL is the main transport

can generate other highly reactive and oxidizing radicals
like lipid-peroxyl (LOO•), oxyl (LO•), and epoxy-allylic
peroxyl (OLOO•). All these radicals, in turn, can lead tovehicle for circulating lipid hydroperoxides (LOOH) andsuggested that HDL lipids are more rapidly oxidized than34)
those in LDL . Some reports show the involvement of oxi-

peroxidation of adjacent lipids; therefore, initiating a chain
52)
peroxidation reaction . The mechanism of such free radi-

dized HDL (OxHDL) in the pathogenesis of various dis35‒38)

eases

.

cal derived oxidation of lipid molecules has been well
49,51,53)

reviewed

Oxidized Lipids. The resultant eﬀects of LPO products arecomplex with diverse structural variability. Increase in the

Oxidation of lipoproteins generates a complex mixture of

number of unsaturated carbons in the lipid generate more
54)
complex mixture of oxidized lipids . Since LOOH are pri-bioactive lipids and accumulating evidence showed thatthey have profound implications in the atherogenesis. Oxi-

mary products of oxidative modiﬁcation, its demonstration

dized lipids commonly refer to lipid derivatives in whichin the lipoproteins can be considered as an early marker ofoxygen is incorporated with the lipid molecules throughlipid oxidation . Importantly, LOOH may also oxidize

55)

5June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Fig. 2.

Structure of lipoprotein and lipid hydroperoxides.

tive evaluation of LOOH in the biological samples haveapolipoproteins, making it unrecognizable by its receptors
56)
and disrupting its metabolic function . It is well-known

been largely limited, because of structural variability andinstability, rapid clearance from circulation, too low con-that oxidative modiﬁcation of lipids and inﬂammation are

centration for easy detection, and the lack of appropriate

major hallmarks in atherosclerosis. These oxidized lipids
play a crucial role to initiate inﬂammatory responses in ath-internal standards for identiﬁcation and quantiﬁcation.
Challenges and precautions in the analysis of lipid oxida-

erosclerosis by interacting with macrophage and endothe. Therefore, oxidized lipid in the lipoprotein istion products have been beautifully reviewed . Thiobarbi-

one of the key promoting factors for its atherogenicity.

turic acid reactive substances (TBARS) assay is perhaps the

Although oxidized lipids are of great interest in oxidative
stress, inﬂammation, and atherosclerosis, they are also asso-

most frequently used markers as an indirect reﬂection of
62)
LPO . Grintzalis et al. have developed a simple spectro-

ciated with number of other pathological conditions likemetric method for the simultaneous measurement of plasma

aging, neurodegenerative diseases including Alzheimer s,malondialdehyde and total LOOH based on the reaction of＋63)
Fe (3 ) with xylenol orange . However, it has low speci-

57,58)

lial cells

61)

Parkinson s, and amyotrophic lateral sclerosis (ALS),
pre-eclampsia and foetal vascular dysfunction, diabetes andﬁcity, as it detects a wide group of aldehydes and alcohols

its long-term complications, chronic kidney diseases,
non-alcoholic fatty liver diseases and steatohepatitis, andpresent in the sample. Similarly, monitoring the formationof conjugated dienes by measuring optical absorbance can59)various cancers .

provide information about the oxidative change in the lipids
but it is largely limited to monitor oxidative change during

Analytical Consideration in Assessing Lipid
Peroxidation

artiﬁcial oxidation of lipids. Likewise, the colorimetric64)
assays by ferrous oxidation using xylenol orange , iodo-

Since LOOH are the major reaction products of lipid oxi-

65)

66)

metric method , ﬂuorometric method

dation, its detection in plasma or lipoprotein fraction is anhave been used forthe detection of total LOOH and reported measurable

60)

indicator of the oxidative change . Despite its importanceamount of lipid peroxides in lipoproteins. More speciﬁc
methods using high-performance liquid chromatography

in the atherosclerotic process, the qualitative and quantita6

June 2020

Medical Mass Spectrometry Vol. 4 No. 1(HPLC) with post-column detection based on ﬂuorome69‒71)

67,68)

try, chemiluminometry

magnetic ﬁeld based on their m/z. Quadrupole time of ﬂight(Q-TOF), Orbitrap MS, and Fourier-transform ion cyclotron

72,73)and electrochemistry

have been reported for the quantiﬁcation of CEOOH, phosphatidylcholine hydroperoxides (PCOOH) and TG hydrop-

resonance (FT-ICR) are commonly used for the identiﬁcation of oxidized lipids to obtain high-resolution mass spec-

eroxides (TGOOH). Using HPLC, we previously reported

tra with mass accuracy within a few ppm of the theoretical
80)
value . MALDI-MS has several important advantages andthat normal young human plasma contains 189±87 nM of74)
CEOOH but no TGOOH . In contract, advanced liver dis-

useful for routine analysis with detailed insights into thesample on a molecular level. The use of MALDI-TOF MS

ease is associated with signiﬁcant elevation of both
CEOOH and TGOOH. PCOOH has been demonstrated inhuman plasma ranging from 0.01 to 0.5 μM using chemilu-

in the analysis of lipid research has been excellently
81)

reviewed by Fuchs, et al . For the quantiﬁcation of
LOOH, triple quadruple MS using selective reaction moni-

minescence HPLC. PCOOH concentration is signiﬁcantly

toring (SRM) or multi reaction monitoring (MRM) should
be used by selecting targeted parent ion in Q1, collision-in-

elevated in diseased condition like diabetes, and it is mainly
75)

carried in LDL and VLDL . Simpler approach for theevaluation of LPO can be the analysis of fatty acid hydrop-

duced dissociations (CID) in Q2, and monitoring the char-eroxides (FAOOH) in the plasma or lipoprotein fraction

acteristic fragments of the parent ion in Q3. Either stable

after saponiﬁcation. Browne et al. previously reported up to
13 distinct regioisomers of FAOOH on oxidatively modi-

isotope (deuterium, 13C or 15N) labeled or lipids with odd
chain FA (generally C17 or C19) are used as internal stan-ﬁed human plasma using HPLC . However, total lipid was

dards in such quantiﬁcation. High-resolution chromato-

extracted from plasma, therefore the source of FAOOH,

graphic separation of lipid and oxidized lipid molecules iswhether it is derived from PL, CE, TG or free FA cannot be

another important aspect. Although GC is routinely used todetermined. Also, the chemical saponiﬁcation using strong

separate volatile compounds in the gas phase, the use of LC

alkali itself can increase the instability of LOOH.

for the separation of lipids has been increasing . Since

76)

82)

The utilization of mass spectrometry (MS) coupled withlipid oxidation products are more hydrophilic than originalgas chromatography (GC) or liquid chromatography (LC)
has revolutionized the study of lipidomic and oxidized lip-

lipids, the use of LC is more suitable tool for the analysis of80)
oxidized lipids than conventional GC-MS . ESI-MS is

ids as it can selectively identify individual or group of compounds. This analytical tool has distinct advantages of sen-most commonly used in the lipidomic study of lipoproteins

sitivity and speciﬁcity with the ability to identify andand is very sensitive for the detection of thermally unstable
83)
LOOH . Mass spectra can be obtained without prior deri-quantify numerous analytes simultaneously based on theirvatization and by direct LC/MS with a few preanalyticalmass to charge (m/z) ratio. Recent progress in lipidomic insteps . Quality of sample is another important issue in thehealth and disease, with a primary focus on analytical77)
approaches, has been recently reviewed . With the devel-

LOOH measurement in lipoproteins. Since LOOH can also
be generated from the auto-oxidation, every eﬀort should

opment of MS-based lipidomics, hundreds of lipid oxida-

be considered to prevent it during sample preparation. In

84)

78)

tion products have been identiﬁed . From the past few

our lab, we use EDTA for the collection of plasma as it acts

decades, liquid chromatography/mass spectrometry (LC/MS) has been widely used for the identiﬁcation and quanti-

as an antioxidant by chelating cationic metals. Similarly,
whenever possible use of EDTA-containing buﬀer is desir-ﬁcation of oxidized lipids derived from FA, CE, and PL inable during isolation and processing of lipoproteins frac-various biological samples and food products. Electrosprayionization (ESI), Matrix-assisted laser desorption/ionization

tions. In order to ensure minimal oxidative changes, sample(MALDI) and atmospheric pressure chemical ionization

preparation should be done at below 4°
C and storage
should be at below −80°
C. Repeated freeze-thaw cycles(APCI) are commonly used ionization techniques in themass spectrometric analysis of non-volatile polar lipids

should be avoided whenever possible for the analysis of85)
LOOH . Also, we routinely use 0.005% (w/v) 2,6-di-tert-

including oxidized lipids. Advantages and limitations ofbutyl-p-cresol (BHT; Butylated hydroxytoluene) in acetonitrile as an antioxidant in order to prevent auto-oxidation ofusing various forms of ionization techniques in lipidomic
79)

have been reviewed . After ionization, the detection oflipid during lipid extraction for MS analysis.
We have reported a novel LC/MS approach for the analy-

analytes in MS depends on their movement in an electric or7

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

sis of CEOOH, PCOOH, and TGOOH

86‒88)

example, in PC34:2 two regioisomeric hydroperoxides ispossible, one at C-9 and other at C-13 of linoleic acid.

. Using highly

sensitive LC/LTQ Orbitrap MS, we identiﬁed several
molecular species of CEOOH and TGOOH in artiﬁcial-

These hydroperoxides can be reduced to corresponding. The development ofly-oxidized LDL and HDL
MS based assay for evaluation of LPO has opened a newhydroxides or dehydrated to its corresponding ketone . A
well-known reaction, involving such reduction of FAOOH

door to researchers to gain more insights into its physiolog-

is the formation of truncated PC, which in turn undergofragmentation to the 5-oxovaleric acid ester of 1-palmi-

86,88)

57)

ical and pathophysiological roles in various diseases. A
well-known clinical study PREVENT (Prospective Randomized Evaluation of the Vascular Eﬀects of Norvasc)

toyl-sn-glycero-3-phosphocholine (POVPC) or glutaricacid ester of 1-palmitoyl-sn-glycero-3-phosphocholine

Trial provided strong evidence of the predictive value of(PGPC) . Detail review on the chemistry of phospholipid

LOOH levels in adverse cardiovascular outcomes inoxidation has been publishedpatients with stable CHD and is independent of traditional
89)
risk factors and inﬂammatory markers . A Scientiﬁc State-of OxPL compounds is largely inﬂuenced by the degree ofment from the American Heart Association (AHA) recom-

A huge number of studies have been published providing

mended measuring speciﬁc products of LPO such as LOOH

a plethora of evidence on the biological signiﬁcance of
OxPL in lipoproteins in inﬂammation. OxPL are biologi-

58)

101)

. In general, the formation
102)

unsaturation on acyl chains of PL

which can be valuable in addressing speciﬁc mechanistic.

questions .
The MS-based assay is also valuable in analyzing oxi-cally potent mediators of various cellular processes mediat-dized free FA. Lui et al. have reported a negative ESI-MS

ultimately linked with ASCVD. Thus it is believed that
OxPL is one of the key players in the process of atherogen-90)103‒108). These responses are

ing inﬂammation and responses

method for the comprehensive study of OxFA in biological109‒112). OxPL can interact with several cellular receptors, including scavenger receptors, platelet-activating fac-

91)

samples . Since lipoproteins contain only a trace amountesis

of free FA, free FAOOH will not be discussed in thistor receptors, peroxisome proliferator-activated receptors,
97)
and Toll-like receptors . Thus, OxPL play an important92)

review but have excellently covered by Massey et al .
Instead, hydroperoxides of other major molecular species inrole in many physiological and pathophysiological processes, such as platelet activation, atherosclerosis, apopto-

lipoproteins will be discussed as follow.

Oxidized Phospholipids

113). In addition, the reactive group of
oxPL can covalently bind to proteins resulting in its dys-sis, and ferroptosis

The surface of lipoproteins is covered primarily by phos-

110)

pholipid that is dominated by phosphatidylcholine (PC) andis directly exposed to various ROS during its systemic cir-function and promote atherogenesis. OxPL can be recog-

nized by the scavenger receptors including CD36 resulting
in unregulated uptake of LDL by macrophage, thus initiat-

culation. Furthermore, the sn-2 position of glycerol backbone of PC is often esteriﬁed with a PUFA making it sus-

ing number of events in the cascade of atherogenicity

ceptible to oxidation. Under oxidative stress, such PUFA in
PL can be easily attached by ROS to generate various oxi-including, inﬂammation, endothelial cells injury, smoothmuscle cells proliferation and chemoattraction of mono-

dized phospholipids (OxPL) which have a wide variety ofcytes

biological activities. About 400 diﬀerent types of OxPL
have been identiﬁed applying the powerful tool of untar-

dized LDL is enriched with oxPC which have profoundeﬀects to increase the pro-inﬂammatory function of. Itabe et al. have reported that minimally oxi-

103,114,115)

93)

116)

geted LC/MS techniques . More than 30 types of OxPL
have been identiﬁed in atherosclerotic lesions . PCOOH. Watson et al. have identiﬁed the bioactive OxPL in
minimally oxidized LDL using LC/MS. Such oxidized lip-are one of the oxidation products of PC, and are known to

ids in LDL can participate in atherosclerosis by activating

be involved in the pathogenesis of various diseases such as

endothelial cells to express monocyte speciﬁc adhesion
117)
molecules and chemokines . Lu et al. performed compre-

LDL

94)

atherosclerosis, Alzheimer s disease, Parkinson s disease,multiple sclerosis, rheumatoid arthritis, diabetes, oxidative
95‒100). Number of regioisostress, and chronic alcoholism

hensive metabolomics in human plasma of patients withmeric hydroperoxides of PC formed depends on the number

the patient with MI compared to stable angina and healthy
control which further supports the oxidative role in athero-

CHD and reported that OxPL were signiﬁcantly elevated inof unsaturated hydrocarbons in the PC molecules. For
8

June 2020

Medical Mass Spectrometry Vol. 4 No. 1. In addition, a high level of OxPL is found inized clinical trial of small scale also showed that the ele-

human atherosclerotic lesions suggesting they are actively
119‒122). Ravinvolved in the progression of atherosclerosis

vated OxPL/apoB levels are predictive of secondary
MACE in patients with stable CHD and the risk can be mit-

andi et al. have exhaustively analyzed all the species of
OxPL in the diﬀerent developmental stages of human ath-

igated therapeutically118)

genesis120). Results from the MIRACL (Myo-cardial Ischemia Reduction With Aggressive Cholesterol95)

erosclerotic plaques . Although there was great variability

Lowering) Trial reported a remarkable increase in OxPL/
apoB levels in patients with acute MI in response to ator-

in the proportions of various oxidation products of PL, thelevel of PCOOH is almost similar to other advance oxida-

132)

vastatin. The study suggested that the increase in OxPL/

apoB levels in response to intervention may reﬂect plaquetion products in advanced stage of the plagues. Therefore,
PCOOH can be a suitable target for risk evaluation of ath-

regression and/or stabilization. Recent study by Philippovaand colleagues have reported LC/MS-based assay to quan-erosclerosis. Similarly, POVPC is another proinﬂammatory
OxPL which is believed to play crucial role on the develop-

tify 8 truncated molecular species of OxPL including

ment of atherosclerosis. POVPC exert wide varieties of biological eﬀects and have been identiﬁed in human athero-

POVPC, PGPC, PONPC and PAzPC. ESI was used as ion
source and MS analysis was performed in negative-ion

sclerotic lesions. Cellular studies have provided abundance

mode. Interestingly, a strong correlation was reported

evidences on its association with chronic inﬂammation andbetween individual molecular species of OxPL but there
were weak or no correlation between LC/MS OxPC quanti-

vascular proliferation by inducing monocytes chemotactic
proteins-1, IL-8, p38 mitogen-activated protein kinase, andﬁcation and immunoassays based on monoclonal antibody

123‒125)c-jun N-terminal kinase. It can also induce muscle

against OxPL in lipoproteinscell proliferations and neutrophil activation and contribute. Since LC/MS method

allows the direct quantiﬁcation of individual species of

126,127)

to platelet aggregation

133). Recent study by Yan et al,

OxPL, it is far more superior to immunoassays.

demonstrated that POVPC is responsible for impaired endo-

From the last decade, MS analysis is the method of

thelial function by uncoupling and inhibiting endothelial

choice for the measurement of oxPL. LC/MS methods fornitric oxide synthase (eNOS) and by inducing endothelial

targeted OxPL species have been reported that enable highsensitivity quantitation in clinical and biological sam-cell apoptosis

125). Both POVPC and PGPC in minimally. Soft ionization techniques̶ESI and MALDI are

oxidized LDL can induce the apoptotic signaling via activa-

plestion of acid sphingomyelinase in arterial smooth muscle

most commonly used in the analysis of PL. Although
ESI-MS is commonly used for the analysis of PCOOH, it

128)

cells

. Therefore, oxPL may play an important role in thedevelopment of atherosclerosis and may be considered as a134)

does not provide structural information about the positionof oxidation on the acyl-chain. The negative ion ESI‒MS

potential therapeutic target for atherosclerosis.
PL with PUFA are susceptible to oxidation and can gen-method reported by Hall and Murphy can give structural

erate an antigenic epitope. Antibodies against such PL areassociated with rheumatologic diseases and antiphospho-

information about the position of oxidation on the side135)

chains . Yin et al. have successfully used negative ESI
ion-trap MS for the identiﬁcation and characterization oflipid antibody syndrome (APS). Dominant epitopes in

136)

OxPL in LDL based on CID fragmentation

OxLDL are OxPL and antibodies against OxPL have also
been used to detect OxPL-protein adducts in human. MALDI-MS

137). Accumulating evidence from large-scale clinicalhas been applied to a wide range of OxPL . Stubiger et
al. used a MALDI-TOF MS approach to proﬁle atherogenic

studies show OxPL/apoB has signiﬁcant diagnostic value

PL in human plasma and lipoproteins demonstrated a posi-and can also be targeted for therapeutic intervention. Data.
Some researchers have applied Ag coordination ionspray (CIS)-MS to improve the ionization, particularly for

129)

LDL

138)

tive correlation between oxidized PC and ASCVD＋

from Bruneck study showed that OxPL/apoB and OxPL/
Lp(a) are strongly and signiﬁcantly associated with the139)

the analysis of highly hydrophilic molecules like CE .
Milne and Porter have applied CIS-MS for the identiﬁca-

presence, extent, and development of atherosclerosis and130). Dallas Heart
Study found that elevated OxPL/apo level can predict secare signiﬁcant predictors of CV eventstion of PCOOH and claimed superior to conventional
140)
ESI-MS . MS analysis of OxPL has been extensively

ondary major adverse cardiovascular events (MACE) in
131)
patients with stable CHD . A recently completed random-

134, 141,142)

reviewed9.

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Most targeted approaches for MS analysis are based on aprecursor ion acquisition of m/z 184 (phosphocholine head-injected into the reversed-phase column with mobile phasecomposed of 10 mM aqueous ammonium acetate, acetoni-group) for the detection of PC, and the neutral loss of water

trile, and 2-propanol. ESI-MS analysis was performed
in both positive- and negative-ion mode in LTQ/Orbitrap(−18 Da) for hydroxides and hydrogen peroxide (−34 Da)for hydroperoxides derivatives. A typical way for the identi-＋

MS and PCOOH were detected as [M＋H] and [M＋−ﬁcation of PCOOH using high-resolution MS is shown in
Fig. 3. As shown in Fig. 3, the synthetic PC16:0/18:2-OOH

CH3COO] , respectively. The MS1 data can be obtained in(PC34:2-OOH) eluted at 8.17 min and its corresponding＋
mass spectrum contained the [M＋H] at m/z 818.5904 (ele-and 2 Hz scan speed. The ion spray voltage was set at
5.0 kV, with a scan range of m/z 250‒1000. The trap ﬁll-

masstime was set at 500 ms. Nitrogen was used as sheath gas818.5904). The EIC at m/z 818.5904 for VLDL showed a(set at 50 arbitrary units). The tandem MS data wereacquired using the collision-induced dissociation in ion-trap

mentalcomposition

C44H85O10NP,

theoretical

Fourier transform mode, with resolving power of 60,000

single peak at RT of 8.18. The base peak observed at m/z＋

mode and data-dependent acquisition, which include scans

818.5907 in (b′) were corresponding to the [M＋H] with thesame elemental composition and exact mass as the corre-on the most intense ions in MS2 (collision energy of 35 V)
and MS3 (collision energy of 45 V), respectively. We identi-

sponding ion for the synthetic standard. Thus, the EIC
peaks of (b) can be identiﬁed as PC34:2-OOH. Similarly,

ﬁed several molecular species of PCOOH in artiﬁcially oxidized LDL and HDL. For the quantiﬁcation, selective reac-

the MS2 and MS3 [Fig. 3 (a″) and (a‴)] spectra of the standard show a base peak at m/z 800.5 and m/z 782.6 corre＋

tion monitoring (SRM) mode was used with the targetedparent to product ions at m/z 778.6 → 760.6 (PC33:1-OOH,＋

sponding to [M＋H−H2O] and [M＋H−2H2O] , respec＋
tively. Ion peaks corresponding to [M＋H‒H2O−O] , [M＋＋＋H-FA18:0] , [M＋H-FA18:2-OOH] can also be detectedIS), m/z 790.6 → 772.6 (PC34:2-OOH) and m/z 818.6 →
800.7 (PC34:2-OOH). Our method has relatively higher

supporting its identiﬁcation. In general, the major fragment

sensitivity with the limit of detection and quantiﬁcation ofions of PCOOH observed in MS2 spectra were a result of

0.01 and 0.08, respectively . Recently, we reported 12

the loss of H2O from the hydroperoxide group, FA fromsn-1 carbon and FAOOH from sn-2 carbon. In contrast, themolecular species of PCOOH in human plasma and TRL
based on calculated elemental composition and high-resolu-

fragment ions of PC in MS2 spectra were formed by thenatural loss of the phosphatidylcholine head-group and FA.

tion m/z with mass accuracy within ±5 ppm, fragmentation
144)
pattern in MS2 and MS3, and RT in LC . PC36:4-OOH

In the targeted MS method for OxPL, the most common

was most often detected in VLDL, IDL, and plasma.
PC34:2-OOH, PC36:2-OOH, and PC36:4-OOH were87)

diagnostic scan is for m/z 184.1 of the phosphocholine headdetected in all plasma samples. PC34:2-OOH, PC36:2OOH, PC36:4-OOH, PC38:4-OOH, PC38:6-OOH, andgroup. Hence, scanning for the precursor of m/z 184.1
enables the detection of all PC and OxPL. For instance,

PC40:6-OOH were major PCOOH species detected in TRL.

POVPC and PGPC can be easily identiﬁed by monitoring
ion fragmentation at m/z 594 → 184 and 610 → 184, respec-

Compared to IDL, detection of PCOOH in VLDL is more
frequent with constant detection of PC36:4-OOH andtively whereas PAPC shows characteristic fragmentation atm/z 782 → 184 . Similarly, SOVPC and SGPC show charac-

PC38:6-OOH. The concentration of PCOOH in lipoprotein

teristic fragmentation at m/z 622 → 184 and 638 → 184,

increases with the increase in the degree of oxidation.
Therefore, the measurement of PCOOH reﬂects the degree

respectively.
A couple of years ago, we published LC/MS-based assayof oxidation of lipoproteins. We found that PC withmono-unsaturated FA (PC34:1 and PC36:1) was found least

for the identiﬁcation and quantiﬁcation of PCOOH in
87)human plasma and lipoprotein fractions . We used inhouse synthesized 1-palmitoyl-2-linoleoyl-PC monohydrop-

susceptible to oxidation, while PC with PUFA particularly,
PC34:3, PC36:3, PC 36:5, and PC38:5 were comparatively

and 1-stearoyl-2-linoleoyl-PC
monohydroperoxide (PC36:2-OOH) as standards for identieroxide(PC34:2-OOH)

144)

more susceptible to oxidation

existence of PCOOH in TRL may provide new insight toﬁcation and 1-palmitoyl-2-heptadecenoyl-PC monohydroperoxide (PC33:1-OOH) as internal standard (IS) for
143)

quantiﬁcation. Our demonstration of theunderstand the pathogenicity of TRL. Alterations of TRL
are linked with endothelial dysfunction and considered to145,146). Hence, thebe atherogenic even when LDL-C are low. Lipid extracts from the lipoproteins were10

June 2020

Fig. 3.

Medical Mass Spectrometry Vol. 4 No. 1

Identiﬁcation of PCOOH using LC/LTQ Orbitrap in positive-ion mode: (a) extracted ion (m/z 790.5593) chromatogram of synthetic PC36:2-OOH (40 pmol); (a′) mass spectrum of the peak at RT 8.17 min in (a); (a″) and (a‴) MS2
and MS3 spectra of PC36:2-OOH at m/z 818.5904, respectively (b) extracted ion (m/z 818.5906) chromatogram of
VLDL; (b′) mass spectrum of the peak at RT 8.18 min in (b); (b″) and (b‴) MS2 and MS3 spectra of PC36:2-OOH at
m/z 818.5904 in VLDL, respectively.

11

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

detection of lipid oxidation products in TRL might provide

lesions and hydroxylated FA are associated with plaquea basis for explaining the atherogenicity of TRL .

33)

instability . The study also demonstrated the presence of

PCOOH, therefore, can be potential targets to be used as
biomarkers in the risk stratiﬁcation of ASCVD or may rep-

CEOOH in plasma of atherosclerotic patients at diﬀerent

resent a potential target for therapeutic intervention. Further

associated with a signiﬁcant generation of CEOOH whichclinical studies are required to assess the clinical utility of. Therefore, CEOOH
are the major component of OxLDL and human atheroscle-

40)

stages. Gerry et al. reported that the oxidation of LDL is155)

increases its uptake by macrophage

PCOOH.
In summary, OxPL are potent biologically active compounds in human lipoproteins contributing to the develop-

rotic lesions. CEOOH are biologically active compound

ment of atherosclerosis. Thus, are potential biomarkers for
cardiac risk prediction and therapeutic intervention. Tech-. Harkewicz et al. haveshown that CEOOH are the major biologically active com-

nological advances in the OxPL measurement will certainly

ponents of minimally oxidized LDL, which in turn stimu-

help to better understanding the chemical and biologicallates wide varieties of cellular and molecular processes

properties of these compounds and provide new insights

involved in atherosclerosis . In addition, the accumulationof CEOOH has been well documented in human atheroscle-that can modulate various cellular process associated withthe progression of ASCVD

41)

into its role in the pathophysiology of human diseases.

rotic lesions and the deposition is disease stage-depen-

Oxidized Cholesteryl Esters

39,40)

dent

147‒149). Using LC/MS method, Hutchins et al. reported

that approximately 23% of CE-18:2, 16% of CE-20:4, and
12% of CE-22:6 in human atherosclerotic lesions were oxi-

Several oxidative products of cholesterol and its ester
have been identiﬁed in lipoproteins and human atherosclerotic lesions

156)

157)

. There are a plethora of studies showing. The same group later reported that up to 92% of
119)
the CE PUFA pool is oxidized in atherosclerotic plaques .

dized

an involvement of oxidized sterols and cholesteryl esters in57)
the development of ASCVD . Cholesterol can be enzy-

They identiﬁed several molecular species of CEOOH in thematically oxidized to generate several oxidized cholesterol
metabolites of oxysterols. It is widely accepted that oxys-

embolized material captured by distal protection ﬁlter

terols play a key role in the development of atherosclerosis

interventions. The role of oxidized CE in inﬂammation andand several studies have been published quantifying it in

its importance in ASCVD has been extensively investigatedhuman plasma. Majority of cholesterol carried in theplasma lipoproteins are esteriﬁed with FA to form corre-

and reviewed by Choi et al .
Initially, HPLC-based measurements of CEOOH weresponding CE. CE with linoleic acid (FA18:2) is the mostpopular. Kenar et al. group were ﬁrst for the study of

predominant in human plasma, followed by oleic acid

CEOOH in OxLDL and OxHDL. However, because ofdevices in cardiovascular patients during percutaneous

150‒153)

154)(FA18:1) and palmitic acid (FA16:0)

158). Trace amounts oftechnical limitations of HPLC single species of CEOOH
148)
were included . The development of MS-based assay forCE esteriﬁed with stearic acid (FA18:0), linolenic acidthe analysis of CEOOH has widened its scope and assisted(FA18:3), arachidonic acid (FA20:4) and DHA (FA22:6)
are also present in the lipoproteins. CE are the most abun-

many clinical and biological studies. Hutchin et al. haveused ESI-MS/MS in human plasma and atheroma samples

dant lipid components in LDL and HDL. PUFA in the CE
are also susceptible to free radical-mediated peroxidation.

and reported that CE18:2 is most predominantly oxi157)
dized . Recent studies by Guo et al. found a positive cor-

Peroxidation of CE results in similar types of oxidative
products as PL. However, unlike PL, which is present in theouter surface, CE is in the inner core of lipoproteins. There-

relation of the levels of CEOOH with diﬀerent types offore, these two classes of lipids are presumably exposed to
diﬀerent oxidative environments in vivo. For example, oxi-cholesterol levels by inhibiting its uptake by hepatocytes bydecreasing LDL-receptor contributing to the pathogenesis

dation of CE18:2 initially generates four primary hydroper57)
oxides at 9- and 13- position of linoleic acid . Reduction. Cholesteryl oleate monohydroperoxideof ASCVD(CE18:1-OOH), cholesteryl arachidonate monohydroperox-

or dehydration of these hydroperoxides generates corre-ide CE20:4-OOH were measured in the study using
ESI-MS in positive-ion mode.

ASCVD and CEOOH in lipoprotein can increase plasma

159,160)

sponding hydroxides or ketone, respectively.

Our original method for the analysis of CEOOH in lipo-

CEOOH are the major lipid oxidation products in human12June 2020

Medical Mass Spectrometry Vol. 4 No. 1

continued to support both fasting and non-fasting plasma

proteins and plasma were based on high-resolution mass86)
spectra using LC/LTQ Orbitrap . We used in-house syn-TG as an independent risk factor for ASCVD. Plasma TG

thesized CE18:1-OOH, cholesteryl linoleate monohydroperoxide (CE18:2-OOH), and cholesteryl linolenate monohy-

are predominantly carried by TRL which have density＜
1.019 kg/L and include chylomicrons, VLDL and IDL.

droperoxide (CE18:3-OOH) as standards for equivocal
identiﬁcation. After extraction of lipids from the lipopro-

Accumulating evidence supports the association of TRL

tein, the extracts were injected into a reversed-phase HPLC
column with a mobile phase composed of 10 mmol/L aque-

VLDL are large enough to enter the arterial endothelial

with atherosclerosis and CHD. Although chylomicrons andlayer, during its circulation lipoprotein lipase utilizes their165)
TG forming intermediate-sized TRL . Such TRL can par-ous ammonium acetate and 2-propanol. ESI-MS analysis
was performed in both positive- and negative-ion mode.
＋

ticipate in atherogenesis like LDL. TRL remnants are con-

＋

166)

CEOOH were detected as [M＋NH4] and/or [M＋Na] in−positive-ion mode and as [M＋CH3COO] in negative-ion

sidered as major atherogenic lipoprotein in plasmamode with mass accuracy of 5.0 ppm. We identiﬁed six
molecular species of CEOOH-CE18:1-OOH, CE-18:2-

metabolic changes that it brings, e.g., reduction of HDL orOOH, CE-18:3-OOH, CE-20:4-OOH (arachidonic acid),
CE-20:5-OOH (EPA), CE-22:6-OOH (DHA) in OxLDL andfully elucidated. Therefore, the speciﬁc role of triglycerides

OxHDL. Based on the high-resolution mass spectra, charac-

has long been controversial

However, whether triglycerides itself is causative or theits indirect reﬂection of remnant lipoproteins remains to bein TRL towards the progression of CHD is uncertain and167‒169).

Oxidized lipids in the TRL may be one of the promoting

teristic retention time in LC and MS/MS spectra, we also
161)

.

33)

. We

factors for its atherogenicity . TG contain a signiﬁcant

provided the ﬁrst evidence that CEOOH are present inamount of unsaturated FA mainly, FA18:1 and FA18:2, and

VLDL and IDL in its native form. We conﬁrmed thatare susceptible to oxidation. Oxidized PL and CE were the

CEOOH in plasma are predominantly carried in TRL. In
vitro studies have shown that both VLDL and IDL canmajor focus for many researchers, but less attention is
given to oxidized TG. A number of studies have demon-

induce the formation of foam cells and CEOOH has been
identiﬁed in human atherosclerotic lesions. It can be specu-

strated that VLDL and IDL can contribute to the formationof foam cells, a hallmark of early atherosclerosis and oxi-

lated that perhaps VLDL and IDL are responsible for thedized forms of these lipoproteins have been identiﬁed in

identiﬁed 6 molecular species of CEOOH in TRL

26,162)delivering CEOOH in the atherosclerotic lesions

26,30).

atherosclerotic lesions. In vitro studies have shown that

Therefore, the presence of CEOOH in TRL might support
the possible involvement of these lipoproteins in the athero-

lipolysis of TRL leads to release several potentially toxic

genic process and can be targeted as biomarkers beyond

Therefore, the study of oxidized TG in TRL can be fruitfulcholesterol.

for a deeper understanding of the atherogenicity of TRL.

oxidized FA which can elicit inﬂammatory responses

170,171).

Our laboratory has identiﬁed several molecular species

Because of poor ionization of CEOOH by ESI or APCI,＋
some researchers have used a silver ion (Ag )-based coordi-

of TGOOH in human plasma and lipoproteins. We use
high-resolution ESI-LTQ Orbitrap MS in positive-ion mode

nation ion spray (CIS) ionization technique for its MS anal＋

mass spectra were obtained in Fourier-transform mode. We
used in-house synthesized standards of TGOOH for equivo-

ysis. CEOOH can be easily detected as Ag adducts and a＋

Hock cleavage of the hydroperoxides induced by Ag
causes region-speciﬁc fragmentation enabling the identiﬁcation position of the hydroperoxides. It includes-1-Oleoyl-2-linoleoyl-3palmitoylglycerol monohydroperoxide (TGOOH-18:1/18:2/

lar as OxPL. However, Gonen and co-workers have16:0), 1,2-dioleoyl-3-palmitoylglycerol monohydroperoxide(TGOOH-18:1/18:1/16:0), and triolein monohydroperoxide

172,173)

cal identiﬁcation

139,149,163). Immunoassay
using antibodies directed against oxidized CE are not popu-(TGOOH-18:1/18:1/18:1). TGOOH are detected as the [Mrecently reported immunoassay that can speciﬁcally detectoxidized CE associated with apo AI and apoB-100 lipopro164)

teins in human plasma＋＋NH4] . MS2 and MS3 spectra show of TGOOH show
characteristic fragmentation pattern with peak correspond-

.

＋

＋

ing to [M＋H‒H2O] , [M＋H‒H2O‒O] , and small peak corresponding to natural loss of FA and FA-OOH. For exam-

Oxidized Triacylglycerols

ple, MS2 spectra of the ion at m/z 906.7748 (TG52:3-OOH)

Many epidemiological, clinical and experimental studies
13June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Table 1.

Summary of literature reviews of analysis of oxidized lipids in biological samples

Oxidized form

Detection methods

Phosphatidylcholine
Hydroperoxides

Hydroxides
Epoxides
Aldehydes

Carboxylate

OxPC

Cholesterylester
Hydroperoxides

Keto
Hydroxy
Epoxy
Oxysterol
OxCETG
Hydroperoxides

Specimen

Targeted diseases

HPLC-chemiluminescent, Total PCOOH

Serum

98, 100

HPLC, Total PCOOH
ESI, LC-MS/MS
ESI, LC-MS/MS
ESI LC-MS/MS
ESI, LC-MS/MS
ESI, LC-MS/MS
CIS-MS
ESI, LC-MS/MS
ESI, LC-MS/MS
HPLC-ﬂuorescent
ESI, LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS, ELISA
ESI, LC-MS/MS
ESI, LC-MS/MS
HPLC, ESI LC-MS/MS, Derivatization
ESI LC-MS/MS, MALDI-TOF MS
MALDI-TOF-MS/MS
ESI, LC-MS/MS
ESI LC-MS/MS
ESI, LC-MS/MS
ESI LC-MS/MS, ELISA
ESI, LC-MS/MS
MALDI-TOF-MS/MS
HPLC, ESI LC-MS/MS, Derivatization
EIA, Total
EIA, Total
EIA, Total
EIA, Total

PlasmaSerum
Atheroma
LDL, HDL, Plasma̶
VLDL, IDL̶̶Atheroma
LDL
Atheroma
PlasmaPlasmaPlasmaAtheroma
LDL
HDL
Plasma, VLDL, LDL, HDL
Atheroma
PlasmaPlasmaPlasmaAtheroma
Plasma, VLDL, LDL, HDL
LDLPlasmaPlasmaPlasma/LDL
PlasmaHyperlipidemia,
oxidative stress̶Chronic alcoholism
CVD
Artiﬁcial oxidation
Artiﬁcial oxidation
Artiﬁcial oxidation̶Artiﬁcial oxidation
CVD
Artiﬁcial oxidation
CVD
Stable angina, MI
CVD
ACS, CAD
Atherosclerosis
Artiﬁcial oxidation
̶Hyperlipidemia
CVD
Stable angina, MI
ACS, CAD
CVD
Atherosclerosis
Hyperlipidemia
Artiﬁcial oxidation
CHD
Atherosclerosis
MI
ACS, CAD

HPLC, Total CEOOH
HPLC, Total CEOOH
HPLC, Total CEOOH
ESI LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS
ESI LC-MS/MS
APCI LC-MS, ESI LC-MS
HPLC
EIA

PlasmaLDL
LDL
PlasmaPlasmaAtheroma
VLDL, IDL
LDL, HDL, Plasma
PlasmaPlasmaPlasmaPlasmaPlasmaLDL
Plasma̶̶̶
CVD
CHD, MI
Atherosclerosis
̶
̶
CVD
CVD
CHD, MICVD
CVD
̶

68, 72, 73
148
155
119
159
157
161
86
119
119
159
119
150
155
164

HPLC, Total TGOOH

Plasma̶

ESI LC-MS/MS
ESI LC-MS/MS

VLDL, IDL
LDL, HDL, Plasma̶Artiﬁcial oxidation

CVD

Reference

72, 75, 141
99
95
87
102
144
140
102
95
116
95
118
119
114, 133
175
117
176
138
95
118
133
119
175
138
117
120, 131
130
132
133

63, 64, 65,
67, 68, 71
174
88

Abs. CVD: cardiovascular diseases, MI: myocardial infarction, CHD: coronary heart disease, ACS: acute coronary syndrome, CAD: coronary artery disease.

14June 2020

Medical Mass Spectrometry Vol. 4 No. 1＋
shows peaks at m/z 871.6 corresponding to [M＋H‒H2O]

inﬂammation and atherogenesis. Molecular and structuralby loss of 18 mass units, m/z 855.5 corresponding to ([M＋＋H‒H2O‒O] by the loss of oxygen, m/z 577.48 correspond-

diversity, instability and trace abundance of LOOH arehydroperoxide, (FAOOH 18:2), m/z 615.46 corresponding

challenges by providing sensitive and speciﬁc detection,
characterization, and quantitation of such complex hetero-

major challenges in the analysis of LOOH. However, theavailability of MS-based assay has largely overcome such＋ing to [M＋H-FAOOH18:2] by loss of linoleic acid mono＋

to [M＋H−FA16:0] by loss of palmitic acid (FA16:0), and＋m/z 589.43 corresponding to [M＋H‒H2O−FA18:1] , which

geneous class of oxidized lipids. LC/MS analysis of LOOH
has opened a new door for better understanding its biologi-

deduced from the precursor ion at m/z 871.64 by loss of
FA18:1. In the negative-ion mode, TGOOH can be detected

cal and clinical implications, particularly in inﬂammation−as [M＋CH3COO] . However, the absolute intensity of theand ASCVD. Wide varieties of methods have been appliedin biological samples to identify and quantify lipid oxida-

same concentration of TGOOH standards is about 350
88)
times higher in positive-ion mode . Therefore, for greatertion products in order to determine its relevance in various

sensitivity positive-ion mode is recommended for quantiﬁ-

diseases, which has been summarized in Table 1. So far, thecation of TGOOH.
Using this high-resolution MS, we were able to identify

results of studies of LOOH were exciting and providing
deeper insights into lipid metabolism. LOOH can be con-

. TG50:2-OOH

sidered as an emerging biomarker for risk stratiﬁcation of
CHD. As the incidence of ASCVD continues to rise glob-

174)

11 molecular species of TGOOH in TRL

is the most predominant TGOOH consistently detected in
VLDL, IDL, and plasma. TG52:3-OOH, TG52:4-OOH,

ally, LOOH can be one of the potential candidate biomark-TG54:3-OOH, TG54:4-OOH, TG54:5-OOH were also predominant in TRL. EPA and DHA containing TGOOH-

ers for the primary prevention of ASCVD and can possibly

TG54:6-OOH, TG56:7-OOH were also identiﬁed. AlthoughMore works are needed in both analytical aspects of LOOH

the mean percentage content of TG in IDL is almost half ofand its clinical assessments to add value in the diagnostic

VLDL, we found that the mean concentration of TGOOH isinvestigations of various diseases and predictive value forhigher in IDL compared to that of VLDL.

CHD. The availability of LC/MS assay for LOOH in thebe targeted for therapeutic intervention to reduce the risk.

We conﬁrm that TRL carries several species of TGOOH
but not by LDL and HDL. When TGOOH containing TRL

clinical laboratory will certainly help to generate more dataon its clinical utility and aid on the evidence-based evalua-

are hydrolyzed by lipoprotein lipase, an enzyme anchoredtion LOOH in clinical practice.

to endothelial wall, the arterial walls are constantly exposedto the liberated FAOOH can further elicit inﬂammatory
responses and trigger the process of atherogenesis. There-

Acknowledgments

fore, it appears that signiﬁcant amounts of oxidized FA are
released during lipolysis of TRL and perhaps this phenome-

Promotion of Science KAKENHI Grants (17H0187919,

This work is supported by the Japanese Society for the
18K0743408 and 19K0786109).

non may result in endothelial dysfunction and inﬂamma170)tion

Conﬂict of Interest. Thus, the presence of TGOOH in VLDL and IDL

All authors declare that they have no conﬂict of interest.

might support a possible involvement of TG and TRL in theatherogenic process. The atherogenicity of TRL can be
partly explained by the existence of TGOOH, along with

Reference

CEOOH and PCOOH. Clinical utility of measuring

1) Rifai N: Lipoproteins and apolipoproteins. Composition,

TGOOH in these lipoproteins needs to be investigated formetabolism, and association with coronary heart disease.

risk assessment of CHD and further works are needed toelucidate the association of such oxidized TG in atheroscle-

Arch Pathol Lab Med 110: 694‒701, 1986.
2) Brown M, Goldstein J: A receptor-mediated pathway forcholesterol homeostasis. Science 232: 34‒47, 1986.

rosis.

3) Sniderman AD, De Graaf J, Couture P, Williams K, Kiss

Summary

RS, et al: Regulation of plasma LDL: The apob paradigm.

In summary, it is now widely accepted that LOOH gener-

Clin Sci 118: 333‒339, 2010.

ated by LPO of lipoproteins play a crucial role to induce

4) Eisenberg S: High density lipoprotein metabolism. J Lipid
15

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Res 25: 1017‒1058, 1984.

17) Steinberg D, Witztum JL: Oxidized low-density lipopro-5) Expert Panel on Detection, Evaluation, and Treatment oftein and atherosclerosis. Arterioscler Thromb Vasc Biol
30: 2311‒2316, 2010.

High Blood Cholesterol in Adults: Executive summary of
the third report of the national cholesterol education pro-18) Moore KJ, Tabas I: Macrophages in the pathogenesis ofatherosclerosis. Cell 145: 341‒355, 2011.

gram (NCEP) expert panel on detection, evaluation, andtreatment of high blood cholesterol in adults (Adult Treat-19) Holvoet P, Collen D: Oxidation of low density lipopro-

ment Panel iii). JAMA 285: 2486‒2497, 2001.

teins in the pathogenesis of atherosclerosis. Atherosclerosis 137: S33-S38, 1998.

6) Rosenson RS, Brewer HB Jr, Davidson WS, Fayad ZA,
Fuster V, et al: Cholesterol eﬄux and atheroprotection:20) Mitra S, Goyal T, Mehta JL: Oxidized LDL, LOX-1 andAdvancing the concept of reverse cholesterol transport.

atherosclerosis. Cardiovasc Drugs Ther 25: 419‒429,

Circulation 125: 1905‒1919, 2012.

2011.

7) 2018

AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/21) Yamaguchi Y, Kunitomo M, Haginaka J: Assay methods

AGS/APhA/ASPC/NLA/PCNA Guideline on the Man-of modiﬁed lipoproteins in plasma. J Chromatogr B Ana-

agement of Blood Cholesterol: Executive Summary: A re-lyt Technol Biomed Life Sci 781: 313‒330, 2002.

port of the American College of Cardiology/American22) Itabe H, Ueda M: Measurement of plasma oxidized

Heart Association Task Force on Clinical Practice Guide-low-density lipoprotein and its clinical implications. J

lines. J Am Coll Cardiol 73: 3168‒3209, 2019.

Atheroscler Thromb 14: 1‒11, 20078) Sandhu PL, Musaad SM, Remaley AT, Buehler SS, Strid-23) Wilson PW, Ben-Yehuda O, McNamara J, Massaro J,

er S, et al: Lipoprotein biomarkers and risk of cardiovas-

Witztum J, et al: Autoantibodies to oxidized LDL and car-cular disease: A laboratory medicine best practices

diovascular risk: The Framingham Oﬀspring Study. Ath-(LMBP) systematic review. J Appl Lab Med 1: 214‒229,

erosclerosis 189: 364‒368, 2006.
24) Hartley A, Haskard D, Khamis R: Oxidized LDL and an-

2016.
9) Hansson GK: Inﬂammation, atherosclerosis, and coronary

ti-oxidized LDL antibodies in atherosclerosis̶Novel in-artery disease. N Engl J Med 352: 1685‒1695, 2005.

sights and future directions in diagnosis and therapy.
Trends Cardiovasc Med 29: 22‒26, 2019.

10) Glass CK, Witztum JL: Atherosclerosis. the road ahead.
Cell 104: 503‒516, 2001.

25) Afonso CB, Spickett CM: Lipoproteins as targets and11) Brown MS, Goldstein JL: Lipoprotein metabolism in themarkers of lipoxidation. Redox Biol 23: 101066, 2019.
26) Krauss RM, Lindgren FT, Williams PT, Kelsey SF, Bren-macrophage: Implications for cholesterol deposition inatherosclerosis. Annu Rev Biochem 52: 223‒261, 1983.

sike J, et al: Intermediate-density lipoproteins and pro-12) Steinberg D, Witztum JL: Is the oxidative modiﬁcation

gression of coronary artery disease in hypercholesterolae-

hypothesis relevant to human atherosclerosis? Do the anti-

mic men. Lancet 2: 62‒66, 1987.

oxidant trials conducted to date refute the hypothesis?

27) Mack WJ, Krauss RM, Hodis HN: Lipoprotein subclasses

Circulation 105: 2107‒2111, 2002.

in the Monitored Atherosclerosis Regression Study

13) Steinberg D, Parhasarathy S, Carew TE, Khoo JC, Witz-(MARS). Treatment eﬀects and relation to coronary an-tum JL: Beyond cholesterol―Modiﬁcations of low-densi-

giographic progression. Arterioscler Thromb Vasc Biolty lipoprotein that increase its atherogenicity. N Engl J

16: 697‒704, 1996.

Med 320: 915‒924, 1989.

28) Hodis HN, Mack WJ, Dunn M, Liu C, Liu C, et al: Inter-14) Witztum JL, Steinberg D: Role of oxidized low density li-

mediate-density lipoproteins and progression of carotid

poprotein in atherogenesis. J Clin Investig 88: 1785‒

arterial wall intima-media thickness. Circulation 95:

1792, 1991.

2022‒2026, 1997.

15) Parthasarathy S, Steinberg D, Witztum JL: The role of ox-29) Krauss RM: Atherogenicity of triglyceride-rich lipopro-

idized low-density lipoproteins in the pathogenesis of ath-teins. Am J Cardiol 81: 13b-7b, 1998.

erosclerosis. Annu Rev Med 43: 219‒225, 1992.

30) Rapp JH, Lespine A, Hamilton RL, Colyvas N, Chau-

16) Berliner JA, Heinecke JW: The role of oxidized lipopro-

meton AH, et al: Triglyceride-rich lipoproteins isolated byteins in atherogenesis. Free Radic Biol Med 20: 707‒727,

selected-aﬃnity anti-apolipoprotein B immunosorption

1996.

from human atherosclerotic plaque. Arterioscler Thromb
16June 2020

Medical Mass Spectrometry Vol. 4 No. 1

14: 1767‒1774, 1994.

inﬂammation. Nat Rev Immunol 15: 511‒523, 2015.

31) Yla-Herttuala S, Jaakkola O, Ehnholm C, Tikkanen MJ,43) Serhan CN, Petasis NA: Resolvins and protectins in in-

Solakivi T, et al: Characterization of two lipoproteins con-ﬂammation resolution. Chem Rev 111: 5922‒5943, 2011.

taining apolipoproteins B and E from lesion-free human44) Dalli J, Serhan CN: Immunoresolvents signaling mole-aortic intima. J Lipid Res 29: 563‒572, 1988.

cules at intersection between the brain and immune sys-32) Hollander W, Paddock J, Colombo M: Lipoproteins in hu-

tem. Curr Opin Immunol 50: 48‒54, 2018.

man atherosclerotic vessels. I. Biochemical properties of45) Davies KJA: Oxidative stress, antioxidant defenses, andarterial low density lipoproteins, very low density lipopro-damage removal, repair, and replacement systems.

teins, and high density lipoproteins. Exp Mol Pathol 30:

IUBMB Life 50: 279‒289, 2000.

144‒171, 1979.

46) Zhong H, Yin H: Role of lipid peroxidation derived 4-hy-33) Cohn JS, Marcoux C, Davignon J: Detection, quantiﬁca-

droxynonenal (4-HNE) in cancer: Focusing on mitochon-tion, and characterization of potentially atherogenic tri-dria. Redox Biology 4: 193‒199, 2015.

glyceride-rich remnant lipoproteins. Arterioscler Thromb47) Zhong H, Xiao M, Zarkovic K, Zhu M, Sa R, et al: Mito-

Vasc Biol 19: 2474‒2486, 1999.

chondrial control of apoptosis through modulation of car-34) Bowry VW, Stanley KK, Stocker R: High density lipopro-

diolipin oxidation in hepatocellular carcinoma: A novel

tein is the major carrier of lipid hydroperoxides in humanlink between oxidative stress and cancer. Free Radic Biolblood plasma from fasting donors. Proc Natl Acad Sci

Med 102: 67‒76, 2017.

USA 89: 10316‒10320, 1992.

48) Xiao M, Zhong H, Xia L, Tao Y, Yin H: Pathophysiology35) Matsunaga T, Koyama I, Hokari S, Komoda T: Detectionof mitochondrial lipid oxidation: Role of 4-hydroxynon-of oxidized high-density lipoprotein. J Chromatogr B An-

enal (4-HNE) and other bioactive lipids in mitochondria.

alyt Technol Biomed Life Sci 781: 331‒343, 2002.

Free Radic Biol Med 111: 316‒327, 2017.

36) Soumyarani VS, N. Jayakumari: Oxidized HDL induces49) Yin H, Porter NA: New insights regarding the autoxida-

cytotoxic eﬀects: Implications for atherogenic mecha-tion of polyunsaturated fatty acids. Antioxidants Redox

nism. J Biochem Mol Toxicol 28: 481‒489, 2014.

Signal 7: 170‒184, 2005.

37) Perségol L, Brindisi MC, Rageot D, Pais de Barros JP,50) Frei B: Reactive oxygen species and antioxidant vitamins:

Monier S, et al: Oxidation-induced loss of the ability of

Mechanisms of action. Am J Med 97: 5S-13S; discussion

HDL to counteract the inhibitory eﬀect of oxidized LDL

22S-8S, 1994.

on vasorelaxation. Heart Vessels 30: 845‒849, 2015.

51) Yin H, Xu L, Porter NA: Free radical lipid peroxidation:
Mechanisms and analysis. Chem Rev 111: 5944‒5972,

38) Wu J, He Z, Gao X, Wu F, Ding R, et al: Oxidized
high-density lipoprotein impairs endothelial progenitor

2011.

cells function by activation of CD36-MAPK-TSP-1 path-52) Thomas JP, Kalyanaraman B, Girotti AW: Involvement ofways. Antioxid Redox Signal 22: 308‒324, 2015.

preexisting lipid hydroperoxides in Cu(2＋)-stimulated ox-39) Carpenter KL, Taylor SE, Ballantine JA, Fussell B, Halli-

idation of low-density lipoprotein. Arch Biochem and Bio-

well B, et al: Lipids and oxidised lipids in human athero-

phys 315: 244‒254, 1994.
53) Niki E, Yoshida Y, Saito Y, Noguchi N: Lipid peroxida-

ma and normal aorta. Biochimica Biophys Acta 1167:
121‒130, 1993.

tion: Mechanisms, inhibition, and biological eﬀects. Biochem Biophys Res Commun 338: 668‒676, 2005.

40) Upston JM, Niu X, Brown AJ, Mashima R, Wang H, et al:
Disease stage-dependent accumulation of lipid and pro-54) Davis TA, Gao L, Yin H, Morrow JD, Porter NA: Vivotein oxidation products in human atherosclerosis. Am J

and in vitro lipid peroxidation of arachidonate esters: The

Pathol 160: 701‒710, 2002.

eﬀect of ﬁsh oil ω -3 lipids on product distribution. J Am
Chem Soc 128: 14897‒14904, 2006.

41) Harkewicz R, Hartvigsen K, Almazan F, Dennis EA,
Witztum JL, et al: Cholesteryl ester hydroperoxides are55) Ayala A, Munoz MF, Arguelles S: Lipid peroxidation:

biologically active components of minimally oxidized

Production, metabolism, and signaling mechanisms oflow density lipoprotein. J Biol Chem 283: 10241‒10251,

malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med

2008.

Cell Longev 2014: 360438, 2014.
56) Kawai Y, Fujii H, Kato Y, Kodama M, Naito M, et al: Es-42) Dennis EA, Norris PC: Eicosanoid storm in infection and17June 2020

Medical Mass Spectrometry Vol. 4 No. 1

teriﬁed lipid hydroperoxide-derived modiﬁcation of pro-tion of hydroperoxides of phosphatidylcholine, cholester-

tein: Formation of a carboxyalkylamide-type lysine ad-ol esters and triacylglycerols by column-switching

duct in human atherosclerotic lesions. Biochem Biophys

high-performance liquid chromatography with a post-col-Res Commun 313: 271‒276, 2004.

umn detection system. J Chromatogr 622: 153‒159,

57) Zhong S, Li L, Shen X, Li Q, Xu W, et al: An update on

1993.

lipid oxidation and inﬂammation in cardiovascular diseas-69) Yamamoto Y: Chemiluminescence-based high-perfor-

es. Free Radic Biol Med 144: 266‒278, 2019.

mance liquid chromatography assay of lipid hydroperox-58) Gianazza E, Brioschi M, Fernandez AM, Banﬁ C: Lipoxi-

ides. Methods Enzymol 233: 319‒324, 1994.
70) Yamamoto Y, Brodsky MH, Baker JC, Ames BN: Detec-

dation in cardiovascular diseases. Redox Biol 23: 101119,

tion and characterization of lipid hydroperoxides at pico-

2019.
59) Negre-Salvayre A, Auge N, Ayala V, Basaga H, Boada J,

mole levels by high-performance liquid chromatography.
Anal Biochem 160: 7‒13, 1987.

et al: Pathological aspects of lipid peroxidation. Free
Radic Res 44: 1125‒1171, 2010.

71) Frei B, Yamamoto Y, Niclas D, Ames BN: Evaluation of60) Girotti AW: Lipid hydroperoxide generation, turnover,

an isoluminol chemiluminescence assay for the detectionand eﬀector action in biological systems. J Lipid Res 9:of hydroperoxides in human blood plasma. Anal Biochem

1529‒1542, 1998.

175: 120‒130, 1988.

61) Schaich KM: Challenges in analyzing lipid oxidation: Are

72) Korytowski W, Bachowski GJ, Girotti AW: Analysis of

one product and one sample concentration enough?, incholesterol and phospholipid hydroperoxides by high-per-Logan A, et al (eds): Lipid oxidation. p. 53‒128, AOCS

formance liquid chromatography with mercury drop elec-

Press, Illinois, 2013.

trochemical detection. Anal Biochem 213: 111‒119, 1993.

62) Tsikas D: Assessment of lipid peroxidation by measuring73) Arai H, Terao J, Abdalla DS, Suzuki T, Takama K: Coulo-

malondialdehyde (MDA) and relatives in biological sam-metric detection in high-performance liquid chromato-ples: Analytical and biological challenges. Anal Biochem

graphic analysis of cholesteryl ester hydroperoxides. Free

524: 13‒30, 2017.

Radic Biol Med 20: 365‒371, 1996.

63) Grintzalis K, Zisimopoulos D, Grune T, Weber D, Geor-

74) Hui SP, Murai T, Yoshimura T, Chiba H, Nagasaka H, etgiou CD: Method for the simultaneous determination of

al: Improved HPLC assay for lipid peroxides in human

free/protein malondialdehyde and lipid/protein hydroper-

plasma using the internal standard of hydroperoxide. Lip-oxides. Free Radic Biol Med 59: 27‒35, 2013.

ids 40: 515‒522, 2005.

64) Nourooz-Zadeh J, Tajaddini-Sarmadi J, Wolﬀ SP: Mea-

75) Miyazawa T: Determination of phospholipid hydroperox-

surement of plasma hydroperoxide concentrations by theides in human blood plasma by a chemilumines-

ferrous oxidation-xylenol orange assay in conjunction

cence-HPLC assay. Free Radic Biol Med 7: 209‒217,

with triphenylphosphine. Anal Biochem 220: 403‒409,

1989.
76) Browne RW, Armstrong D: HPLC analysis of lipid-de-

1994.
65) Cramer GL, Miller JF, Pendleton RB, Lands WE: Iodo-

rived polyunsaturated fatty acid peroxidation products inmetric measurement of lipid hydroperoxides in humanoxidatively modiﬁed human plasma. Clin Chem 46: 829‒

plasma. Anal Biochem 193: 204‒211, 1991.

836, 2000.

66) Santas J, Guzmán YJ, Guardiola F, Rafecas M, Bou R:77) Wei F, Lamichhane S, Orešič M, Hyötyläinen T: Lipi-

High-throughput analysis of lipid hydroperoxides in edi-

domes in health and disease: Analytical strategies andble oils and fats using the ﬂuorescent reagent diphe-

considerations. Trends in Analytical Chemistry 120:nyl-1-pyrenylphosphine. Food Chem 162: 235‒241, 2014.

115664, 2019.

67) Akasaka K, Ohrui H, Meguro H, Tamura M: Determina-78) Norris PC, Dennis EA: A lipidomic perspective on inﬂam-

tion of triacylglycerol and cholesterol ester hydroperox-

matory macrophage eicosanoid signaling. Adv Biol Regul

ides in human plasma by high-performance liquid chro-

54: 99‒110, 2014.

matography with ﬂuorometric post column detection. J79) Murphy RC, Leiker TJ, Barkley RM: Glycerolipid andChromatogr 617: 205‒211, 1993.

cholesterol ester analyses in biological samples by mass

68) Akasaka K, Ohrui H, Meguro H: Simultaneous determina-

spectrometry. Biochim Biophys Acta 1811: 776‒783,
18

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

PREVENT study. J Am Coll Cardiol 51: 1196‒1202,

2011.
80) Li L, Zhong S, Shen X, Li Q, Xu W, et al: Recent devel-

2008.

opment on liquid chromatography-mass spectrometry90) Griendling KK, Touyz RM, Zweier JL, Dikalov S, Chil-analysis of oxidized lipids. Free Radic Biol Med 144: 16‒

ian W, et al: Measurement of reactive oxygen species, re-34, 2019.

active nitrogen species, and redox-dependent signaling in81) Fuchs B, Süss R, Schiller J: An update of MALDI-TOF

the cardiovascular system: A scientiﬁc statement from themass spectrometry in lipid research. Prog Lipid Res 9:

American heart association. Circ Res 119: e39‒75, 2016.

450‒475, 2010.

91) Liu X, Moon SH, Mancuso DJ, Jenkins CM, Guan S, et82) Bajoub A, Pacchiarotta T, Hurtado-Fernández E, Ol-

al: Oxidized fatty acid analysis by charge-switch derivat-

mo-García L, García-Villalba R, et al: Comparing two

ization, selected reaction monitoring, and accurate mass

metabolic proﬁling approaches (liquid chromatography

quantitation. Anal Biochem 442: 40‒50, 2013.

and gas chromatography coupled to mass spectrometry)92) Massey KA, Nicolaou A: Lipidomics of oxidized polyun-for extra-virgin olive oil phenolic compounds analysis: A

saturated fatty acids. Free Radic Biol Med 59: 45‒55,

botanical classiﬁcation perspective. J Chromatogr A

2013.

1428: 267‒279, 2016.

93) Aoyagi R, Ikeda K, Isobe Y, Arita M: Comprehensive83) Scherer M, Böttcher A, Liebisch G: Lipid proﬁling of li-

analyses of oxidized phospholipids using a measured MS/

poproteins by electrospray ionization tandem mass spec-

MS spectra library. J Lipid Res 58: 2229‒2237, 2017.

trometry. Biochim Biophys Acta 1811: 918‒924, 2011.

94) Marathe GK, Zimmerman GA, Prescott SM, McIntyre84) MacMillan DK, Murphy RC: Analysis of lipid hydroper-

TM: Activation of vascular cells by PAF-like lipids in oxi-oxides and long-chain conjugated keto acids by negative

dized LDL. Vasc Pharmacol 38: 193‒200, 2002.

ion electrospray mass spectrometry. J Am Soc Mass Spec-95) Ravandi A, Babaei S, Leung R, Monge JC, Hoppe G, ettrom 6: 1190‒1201, 1995.

al: Phospholipids and oxophospholipids in atherosclerotic85) Ferreiro-Vera C, Ribeiro JP, Mata-Granados JM, Priego-

plaques at diﬀerent stages of plaque development. Lipids

Capote F, Luque de Castro MD: Standard operation proto-

39: 97‒109, 2004.

col for analysis of lipid hydroperoxides in human serum

96) Leitinger N: The role of phospholipid oxidation products

using a fully automated method based on solid-phase ex-in inﬂammatory and autoimmune diseases: Evidence fromtraction and liquid chromatography-mass spectrometry inanimal models and in humans. Subcell Biochem 49: 325‒

selected reaction monitoring. J Chromatogr A 1218:

350, 2008.

6720‒6726, 2011.

97) Greig FH, Kennedy S, Spickett CM: Physiological eﬀects86) Hui SP, Sakurai T, Ohkawa F, Furumaki H, Jin S, et al:of oxidized phospholipids and their cellular signaling

Detection and characterization of cholesteryl ester hy-

mechanisms in inﬂammation. Free Radic Biol Med 52:

droperoxides in oxidized LDL and oxidized HDL by use

266‒280, 2012.
98) Kinoshita M, Oikawa S, Hayasaka K, Sekikawa A, Na-of an Orbitrap mass spectrometer. Anal Bioanal Chem
404: 101‒112, 2012.

gashima T, et al: Age-related increases in plasma phospha-87) Hui SP, Taguchi Y, Takeda S, Ohkawa F, Sakurai T, et al:tidylcholine hydroperoxide concentrations in control sub-Quantitative determination of phosphatidylcholine hy-

jects and patients with hyperlipidemia. Clin Chem 46:

droperoxides during copper oxidation of LDL and HDL

822‒828, 2000.

by liquid chromatography/mass spectrometry. Anal Bio-99) Yang L, Latchoumycandane C, McMullen MR, Pratt BT,

anal Chem 403: 1831‒1840, 2012.

Zhang R, et al: Chronic alcohol exposure increases circu-

88) Hui SP, Sakurai T, Takeda S, Jin S, Fuda H, et al: Analy-

lating bioactive oxidized phospholipids. J Biol Chem 285:

sis of triacylglycerol hydroperoxides in human lipopro-

22211‒22220, 2010.

teins by Orbitrap mass spectrometer. Anal Bioanal Chem

100) Adachi J, Matsushita S, Yoshioka N, Funae R, Fujita T, et405: 4981‒4987, 2013.

al: Plasma phosphatidylcholine hydroperoxide as a new89) Walter MF, Jacob RF, Bjork RE, Jeﬀers B, Buch J, et al:marker of oxidative stress in alcoholic patients. J Lipid
Res 45: 967‒971, 2004.

Circulating lipid hydroperoxides predict cardiovascular101) Reis A, Spickett CM: Chemistry of phospholipid oxida-

events in patients with stable coronary artery disease: The
19

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

tion. Biochim Biophys Acta 1818: 2374‒87, 2012.

oxidized phosphatidylcholines. J Biochem 134: 459‒465,102) Khoury S, Pouyet C, Lyan B, Pujos-Guillot E: Evaluation

2003.

of oxidized phospholipids analysis by LC-MS/MS. Anal

117) Watson AD, Leitinger N, Navab M, Faull KF, Hörkkö S,

Bioanal Chem 410: 633‒647, 2018.

et al: Structural identiﬁcation by mass spectrometry of ox-103) Miller YI, Shyy JYJ: Context-dependent role of oxidized

idized phospholipids in minimally oxidized low density

lipids and lipoproteins in inﬂammation. Trends Endocri-

lipoprotein that induce monocyte: Endothelial interactions

nol Metabol 28: 143‒152, 2017.

and evidence for their presence in vivo. J Biol Chem 272:
13597‒13607, 1997.

104) Romanoski CE, Che N, Yin F, Mai N, Pouldar D, et al:
Network for activation of human endothelial cells by oxi-118) Lu J, Chen B, Chen T, Guo S, Xue X, et al: Comprehen-dized phospholipids: A critical role of heme oxygenase 1.

sive metabolomics identiﬁed lipid peroxidation as a prom-

Circ Res 109: e27‒41, 2011.

inent feature in human plasma of patients with coronary105) Bretscher P, Egger J, Shamshiev A, Trötzmüller M, Köfel-heart diseases. Redox Biol 12: 899‒907, 2017.

er H, et al: Phospholipid oxidation generates potent an-119) Ravandi A, Leibundgut G, Hung MY, Patel M, Hutchins

ti-inﬂammatory lipid mediators that mimic structurally re-PM, et al: Release and capture of bioactive oxidized phos-

lated pro-resolving eicosanoids by activating Nrf2. EMBO

pholipids and oxidized cholesteryl esters during percuta-

Mol Med 7: 593‒607, 2015.

neous coronary and peripheral arterial interventions in hu-106) Thomas CP, O Donnell VB: Oxidized phospholipid sig-mans. J Am Coll Cardiol 63: 1961‒1971, 2014.

naling in immune cells. Curr Opin Pharmacol 12: 471‒

120) Byun YS, Lee JH, Arsenault BJ, Yang X, Bao W, et al:

477, 2012.

Relationship of oxidized phospholipids on apolipoprotein

107) Freigang S: The regulation of inﬂammation by oxidized

B-100 to cardiovascular outcomes in patients treated withphospholipids. Eur J Immunol 46: 1818‒1825, 2016.

intensive versus moderate atorvastatin therapy: The TNT
trial. J Am Coll Cardiol 65: 1286‒1295, 2015.

108) Aldrovandi M, O Donnell VB: Oxidized PLs and vascularinﬂammation. Curr Atheroscler Rep 15: 323, 2013.

121) Capoulade R, Chan KL, Yeang C, Mathieu P, Bossé Y, et
al: Oxidized phospholipids, lipoprotein(a), and progres-109) Salomon RG: Structural identiﬁcation and cardiovascular
activities of oxidized phospholipids. Circ Res 111: 930‒

sion of calciﬁc aortic valve stenosis. J Am Coll Cardiol

946, 2012.

66: 1236‒1246, 2015.

110) Berliner JA, Watson AD: A role for oxidized phospholip-122) Berliner JA, Subbanagounder G, Leitinger N, Watson AD,

ids in atherosclerosis. N Engl J Med 353: 9‒11, 2005.

Vora D: Evidence for a role of phospholipid oxidation

111) Berliner JA, Leitinger N, Tsimikas S: The role of oxidized

products in atherogenesis. Trends Cardiovasc Med 11:
142‒147, 2001.

phospholipids in atherosclerosis. J Lipid Res 50:
S207-S212, 2009.

123) Fruhwirth GO, Moumtzi A, Loidl A, Ingolic E, Hermetter

112) Lee S, Birukov KG, Romanoski CE, Springstead JR, Lu-

A: The oxidized phospholipids POVPC and PGPC inhibit

sis AJ, et al: Role of phospholipid oxidation products ingrowth and induce apoptosis in vascular smooth muscle

atherosclerosis. Circ Res 111: 778‒799, 2012.

cells. Biochim Biophys Acta 1761: 1060‒1069, 2006.

113) Li PL, Gulbins E: Bioactive lipids and redox signaling:124) Pégorier S, Stengel D, Durand H, Croset M, Ninio E: Oxi-

Molecular mechanism and disease pathogenesis. Antioxi-

dized phospholipid: POVPC binds to platelet-activat-

dants Redox Signal 28: 911‒915, 2018.

ing-factor receptor on human macrophages. Implications

114) Fruhwirth GO, Loidl A, Hermetter A: Oxidized phospho-

in atherosclerosis. Atherosclerosis 188: 433‒443, 2006.
125) Yan FX, Li HM, Li SX, He SH, Dai WP, et al: The oxi-lipids: from molecular properties to disease. Biochim Biophys Acta 1772: 718‒736, 2007.

dized phospholipid POVPC impairs endothelial function115) Serbulea V, DeWeese D, Leitinger N: The eﬀect of oxi-and vasodilation via uncoupling endothelial nitric oxide

dized phospholipids on phenotypic polarization and func-

synthase. J Mol Cell Cardiol 112: 40‒48, 2017.

tion of macrophages. Free Radic Biol Med 111: 156‒168,126) Heery JM, Kozak M, Staﬀorini DM, Jones DA, Zimmerman GA, et al: Oxidatively modiﬁed LDL contains phos-2017.
116) Itabe H, Mori M, Fujimoto Y, Higashi Y, Takano T: Mini-

pholipids with platelet-activating factor-like activity andmally modiﬁed LDL is an oxidized LDL enriched withstimulates the growth of smooth muscle cells. J Clin In20

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

vest 96: 2322‒2330, 1995.

of red blood cell ghosts with t-butylhydroperoxide. Anal
Biochem 258: 184‒194, 1998.

127) Marathe GK, Davies SS, Harrison KA, Silva AR, Murphy
RC, et al: Inﬂammatory platelet-activating factor-like

136) Yin H, Cox BE, Liu W, Porter NA, Morrow JD, et al:phospholipids in oxidized low density lipoproteins are

Identiﬁcation of intact oxidation products of glycerophos-fragmented alkyl phosphatidylcholines. J Biol Chem 274:

pholipids in vitro and in vivo using negative ion electro-

28395‒28404, 1999.

spray iontrap mass spectrometry. J Mass Spectrom 44:128) Loidl A, Sevcsik E, Riesenhuber G, Deigner HP, Hermet-

672‒680, 2009.

ter A: Oxidized phospholipids in minimally modiﬁed low137) Spickett CM, Pitt AR: Oxidative lipidomics coming of

density lipoprotein induce apoptotic signaling via activa-

age: Advances in analysis of oxidized phospholipids intion of acid sphingomyelinase in arterial smooth muscle

physiology and pathology. Antioxid Redox Signal 22:

cells. J Biol Chem 278: 32921‒32928, 2003.

1646‒1666, 2015.

129) Horkko S, Bird DA, Miller E, Itabe H, Leitinger N, et al:138) Stubiger G, Aldover-Macasaet E, Bicker W, Sobal G,

Monoclonal autoantibodies speciﬁc for oxidized phospho-

Willfort-Ehringer A, et al: Targeted proﬁling of atherogen-lipids or oxidized phospholipid-protein adducts inhibit

ic phospholipids in human plasma and lipoproteins of hy-

macrophage uptake of oxidized low-density lipoproteins.

perlipidemic patients using MALDI-QIT-TOF-MS/MS.

J Clin Invest 103: 117‒128, 1999.

Atherosclerosis 224: 177‒186, 2012.

130) Tsimikas S, Kiechl S, Willeit J, Mayr M, Miller ER, et al:139) Yin H, Porter NA: Identiﬁcation of intact lipid peroxides

Oxidized phospholipids predict the presence and progres-by Ag coordination ion-spray mass spectrometry (CIS-sion of carotid and femoral atherosclerosis and symptom-

MS). Methods Enzymol 433: 193‒211, 2007.

＋

atic cardiovascular disease: Five-year prospective results140) Milne GL, Porter NA: Separation and identiﬁcation of

from the Bruneck study. J Am Coll Cardiol 47: 2219‒

phospholipid peroxidation products. Lipids 36: 1265‒

2228, 2006.

1275, 2001.

131) Tsimikas S, Clopton P, Brilakis ES, Marcovina SM,141) Domingues MR, Reis A, Domingues P: Mass spectrome-

Khera A, et al: Relationship of oxidized phospholipids on

try analysis of oxidized phospholipids. Chem Phys Lipids

apolipoprotein B-100 particles to race/ethnicity, apolipo-

156: 1‒12, 2008.

protein(a) isoform size, and cardiovascular risk factors.

142) Reis A: Oxidative Phospholipidomics in health and dis-Results from the Dallas heart study. Circulation 108:

ease: Achievements, challenges and hopes. Free Radic

836940, 2009.

Biol Med 111: 25‒37, 2017.

132) Fraley AE, Schwartz GG, Olsson AG, Kinlay S, Szarek143) Hui SP, Chiba H, Sakurai T, Asakawa C, Nagasaka H, et

M, et al: Relationship of oxidized phospholipids and bio-al. An improved HPLC assay for phosphatidylcholine hy-

markers of oxidized low-density lipoprotein with cardio-

droperoxides (PCOOH) in human plasma with synthetic

vascular risk factors, inﬂammatory biomarkers, and eﬀect

PCOOH as internal standard. J Chromatogr B Analyt

of statin therapy in patients with acute coronary syn-Technol Biomed Life Sci 857: 158‒163, 2007.

dromes: Results from the MIRACL (Myocardial Ischemia

144) Shrestha R, Chen Z, Miura Y, Yamamoto Y, Sakurai T, etReduction With Aggressive Cholesterol Lowering) trial. J

al: Identiﬁcation of molecular species of phosphatidylcho-

Am Coll Cardiol 53: 2186‒2196, 2009.

line hydroperoxides in native and copper-oxidized triglyceride-rich lipoproteins in humans. Ann Clin Biochem

133) Philippova M, Oskolkova OV, Bicker W, Schoenenberger

57: 95‒98, 2020.

AW, Resink TJ, et al: Analysis of fragmented oxidized
phosphatidylcholines in human plasma using mass spec-145) Lucero D, López GI, Gorzalczany S, Duarte M, González

trometry: Comparison with immune assays. Free RadicBE, et al: Alterations in triglyceride rich lipoproteins are

Biol Med 144: 167‒175, 2019.

related to endothelial dysfunction in metabolic syndrome.
Clin Biochem 49: 932‒935, 2016.

134) O Donnell VB: Mass spectrometry analysis of oxidized
phosphatidylcholine and phosphatidylethanolamine. Bio-146) Lawler PR, Akinkuolie AO, Chu AY, Shah SH, Kraus
WE, et al: Atherogenic lipoprotein determinants of cardio-

chim Biophys Acta 1811: 818‒826, 2011.
135) Hall LM, Murphy RC: Analysis of stable oxidized molec-

vascular disease and residual risk among individuals withular species of glycerophospholipids following treatment

low low-density lipoprotein cholesterol. J Am Heart Assoc
21

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

erosclerosis. Mol Aspects Med 24: 239‒250, 2003.

6: e005549, 2017.
147) Yin H, Havrilla CM, Morrow JD, Porter NA: Formation

157) Hutchins PM, Moore EE, Murphy RC: Electrospray MS/

of isoprostane bicyclic endoperoxides from the autoxida-

MS reveals extensive and nonspeciﬁc oxidation of choles-

tion of cholesteryl arachidonate. J Am Chem Soc 124:

terol esters in human peripheral vascular lesions. J Lipid

7745‒7754, 2002.

Res 52: 2070‒2083, 2011.

148) Kenar JA, Havrilla CM, Porter NA, Guyton JR, Brown

158) Choi SH, Sviridov D, Miller YI: Oxidized cholesteryl es-

SA, et al: Identiﬁcation and quantiﬁcation of the regioiso-

ters and inﬂammation. Biochim Biophys Acta Mol Cellmeric cholesteryl linoleate hydroperoxides in oxidized hu-

Biol Lipids 1862: 393‒397, 2017.

man low density lipoprotein and high density lipoprotein.

159) Guo S, Lu J, Zhuo Y, Xiao M, Xue X, et al: Endogenous

Chem Res Toxicol 9: 737‒744, 1996.

cholesterol ester hydroperoxides modulate cholesterol lev＋

els and inhibit cholesterol uptake in hepatocytes and mac-

149) Havrilla CM, Hachey DL, Porter NA: Coordination (Ag )ion spray‒mass spectrometry of peroxidation products ofcholesteryllinoleateandcholesterylrophages. Redox Biol 21: 101069, 2019.

arachidonate:160) Guo S, Li L, Yin H: Cholesterol homeostasis and liver X

High-performance lipid chromatography‒mass spectrome-

receptor (LXR) in atherosclerosis. Cardiovasc Haematol

try analysis of peroxide products from polyunsaturated

Disord-Drug Targets 18: 27‒33, 2018.

lipid autoxidation. J Am Chem Soc 122: 8042‒8055,

161) Shrestha R, Hui SP, Sakurai T, Yagi A, Takahashi Y, et al:
Identiﬁcation of molecular species of cholesteryl ester hy-

2000.
150) McDonald JG, Smith DD, Stiles AR, Russell DW: A com-droperoxides in very low-density and intermediate-density

prehensive method for extraction and quantitative analy-

lipoproteins. Ann Clin Biochem 51: 662‒671, 2014.

sis of sterols and secosteroids from human plasma. J Lip-

162) Herijgers N, Van Eck M, Korporaal SJ, Hoogerbrugge

id Res 53: 1399‒1409, 2012.

PM, Van Berkel TJ: Relative importance of LDL receptor151) Liu W, Xu L, Lamberson CR, Merkens LS, Steiner RD, etand scavenger receptor class B in the beta-VLDL-induced

al: Assays of plasma dehydrocholesteryl esters and oxys-uptake and accumulation of cholesteryl esters by peritone-

terols from Smith‒Lemli‒Opitz syndrome patients. J Lip-

al macrophages. J Lipid Res 41: 1163‒1171, 2000.

id Res 54: 244‒253, 2013.

163) Yin H, Hachey DL, Porter NA: Analysis of diacyl perox-152) Griﬃths WJ, Wang Y, Karu K, Samuel E, McDonnell S,

ides by Ag＋coordination ionspray tandem mass spectrom-

et al: Potential of sterol analysis by liquid chromatogra-

etry: Free radical pathways of complex decomposition. J

phy-tandem mass spectrometry for the prenatal diagnosis

Am Soc Mass Spectrom 12: 449‒455, 2001.

of Smith‒Lemli‒Opitz syndrome. Clin Chem 54: 1317‒164) Gonen A, Choi SH, Miu P, Agatisa-Boyle C, Acks D, et

1324, 2008.

al: A monoclonal antibody to assess oxidized cholesteryl153) Griﬃths WJ, Gilmore I, Yutuc E, Abdel-Khalik J, Crickesters associated with apoAI and apoB-100 lipoproteins in

PJ, et al: Identiﬁcation of unusual oxysterols and bile ac-

human plasma. J Lipid Res 60: 436‒445, 2019.

ids with 7-oxo or 3beta,5alpha,6beta-trihydroxy functions165) Nordestgaard BG: A new start for triglycerides and rem-in human plasma by charge-tagging mass spectrometry

nant cholesterol-nonfasting. Clin Chem 63: 1418‒1419,

with multistage fragmentation. J Lipid Res 59: 1058‒

2017.
166) Nakajima K, Nakano T, Tanaka A: The oxidative modiﬁ-

1070, 2018.
154) Miura Y, Furukawa T, Kobayashi M, Shrestha R, Taka-cation hypothesis of atherosclerosis: The comparison ofhashi R, et al: Absolute quantiﬁcation of cholesteryl esters

atherogenic eﬀects on oxidized LDL and remnant lipopro-using liquid chromatography-tandem mass spectrometryteins in plasma. Clin Chim Acta 367: 36‒47, 2006.

uncovers novel diagnostic potential of urinary sediment.

167) Rapaport E, Bilheimer DW, Chobanian AV, Hajjar DP,

Steroids 123: 43‒49, 2017.

Hawkins CM, et al: NIH consensus development panel on155) Gerry AB, Satchell L, Leake DS: A novel method fortriglyceride, high density lipoprotein, and coronary heart

production of lipid hydroperoxide- or oxysterol-richdisease. JAMA 269: 505‒510, 1993.

low-density lipoprotein. Atherosclerosis 197: 579‒587,168) Goldberg IJ, Eckel RH, McPherson R: Triglycerides and2008.

heart disease: Still a hypothesis? Arterioscler Thromb156) Leitinger N: Cholesteryl ester oxidation products in ath-

Vasc Biol 31: 1716‒1725, 2011.
22

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

169) Nordestgaard BG, Varbo A: Triglyceride and cardiovascu-

Simple chemical syntheses of TAG monohydroperoxides.

lar disease. Lancet 384: 626‒635, 2014.

Lipids 38: 1287‒1292, 2003.

170) Wang L, Gill R, Pedersen TL, Higgins LJ, Newman JW,

174) Shrestha R, Hui SP, Miura Y, Yagi A, Takahashi Y, et al:

et al: Triglyceride-rich lipoprotein lipolysis releases neu-

Identiﬁcation of molecular species of oxidized triglycer-

tral and oxidized FFAs that induce endothelial cell inﬂam-

ide in plasma and its distribution in lipoprotein. Clin

mation. J Lipid Res 50: 204‒213, 2009.

Chem Lab Med 53: 1859‒1869, 2015.

171) Saraswathi V, Hasty AH: The role of lipolysis in mediat-175) Podrez EA, Poliakov E, Shen Z, Zhang R, Deng Y, et al:ing the proinﬂammatory eﬀects of very low density lipo-

A novel family of atherogenic oxidized phospholipids

proteins in mouse peritoneal macrophages. J Lipid Res

promotes macrophage foam cell formation via the scaven-

47: 1406‒1415, 2006.

ger receptor CD36 and is enriched in atherosclerotic lesions. J Biol Chem 277: 38517‒38523, 2002.

172) Hui SP, Murai T, Yoshimura T, Chiba H, Nagasaka H, etal: Improve HPLC assay for lipid peroxides in human176) Gao D, Podrez EA: Characterization of covalent modiﬁ-

plasma using the internal standard of hydroperoxide. Lip-

cations of HDL apoproteins by endogenous oxidized

ids 40: 515‒522, 2005.

phospholipids. Free Radic Biol Med 115: 57‒67, 2018.

173) Hui SP, Murai T, Yashimura T, Chiba H, Kurosawa T.

23