Lipid Peroxidation

Lipid peroxidation (LP) is the free radical-mediated cell and tissue injury that forms
lipid peroxides within cell membranes and organelles.

From: Advances in Pharmacology, 1994

Related terms:

Antioxidant, Oxidative Stress, Lipids, Enzymes, Toxicity, Antioxidant Activity, Pro-

teins, Superoxide Dismutase, Vitamin E

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Lipid Peroxidation
Zhengwei Cai, in Encyclopedia of Toxicology (Second Edition), 2005

What is Lipid Peroxidation?

Lipid peroxidation is an oxidative chain reaction in which one lipid molecule after
another becomes oxidized to the maximum possible extent or so as to form a
lipid peroxide (i.e., a lipid molecule containing one or more O–O bonds). At high
temperatures, lipid peroxides decompose to produce a range of unpleasant-tasting
and foul-smelling products such as epoxides, ketones, acids, and aldehydes. Most
biological membranes are extended bilayers of amphiphilic lipids with hydrophobic
moieties directed to the center and hydrophilic head groups at the two surfaces.
Biological cell membranes are packed with polyunsaturated fatty acids (PUFAs), such
as arachidonic and docosahexaenoic acid, in either the isolated form or the incorpo-
rated form in triacylglycerides and phospholipids. PUFAs are particularly susceptible
to peroxidation. With increasing concerns about the potential adverse effects of lipid
peroxidation in cellular membranes, the relevance of lipid peroxidation to biology
and human diseases has been extensively explored since the 1950s.

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Lipid Peroxidation
S.N. Desai, ... S.D. Ray, in Encyclopedia of Toxicology (Third Edition), 2014

Measurement of Lipid Peroxidation

Lipid peroxidation can be determined quantitatively or qualitatively by a variety of
methods. It can be measured by losses of fatty acids; amounts of primary peroxida-
tion products; amounts of secondary products such as carbonyls and hydrocarbon
gases; and reduction in antioxidant activity. Some of the commonly used methods
are described below. Analysis of fatty acids by gas liquid chromatography (GLC) or
high-performance liquid chromatography (HPLC) is used for measuring the loss of
unsaturated fatty acids, a consequence of lipid peroxidation. Lipid hydroperoxides,
the primary product of peroxidation, can be directly measured by HPLC with chemi-
luminescence detectors. Iodine liberation and glutathione peroxidase methods are
often used for measuring lipid peroxides. Lipid peroxides oxidize I− to I2 for titration
with thiosulfate and thus consumption of thiosulfate indirectly indicates the quantity
of lipid peroxides. Hydrogen peroxides and hydroperoxides oxidize reduced GSH to
GSSG. The addition of glutathione reductase and NADPH reduces GSSG back to
GSH, requiring consumption of NADPH, which can be related to peroxide content.
Spin traps (phenyl t-butylnitrone) are frequently used for trapping intermediate
radicals. Products of lipid peroxide decomposition, such as hydrocarbon gases and
cytotoxic aldehydes, can be measured by GLC or HPLC. Lipid peroxidation products
may cause damage to DNA, and formation of 8-oxo-2 -deoxyguanosine (8oxodG) is
a marker of oxidative damage to DNA. Contents of 8oxodG in DNA can be quantified
by HPLC with an EC detector and by an immunochemical method (ELISA).

The most popular assays for measurement of lipid peroxidation are the thiobarbi-
turic acid (TBA) test and diene conjugation determination. In the TBA test, lipid-con-
taining samples are heated with TBA and an LP product, malondialdehyde (MDA) at
low pH to allow the formation of a pink colored complex (see reaction below). Density
of the color is related to the extent of lipid peroxidation. Because of its simplicity and
economy, this method is used extensively in in vivo and in vitro settings. During the
process of lipid peroxidation, diene conjugations (a double bond–single bond–dou-
ble bond structure) are formed (see enzyme-catalyzed peroxidation), which absorb
ultraviolet (UV) light in the wavelength range of 230–235 nm. The absorption of
UV light at this wavelength can be related to contents of diene conjugates in lipid
extracts of tissues and, thus, to extent of lipid peroxidation.
Other newly developed, routinely used, methods are 4-hydroxynonenal assay and
8-iso-prostaglandin F2a assay. Elisa kits are now mostly used to detect various
lipid peroxidation adducts. These kits are readily available on the market, are quite
specific, and give accurate results. Since both MDA and hydroxynonenal (HNE)
have been shown to be capable of binding to proteins and forming stable adducts,
they are also considered advanced lipid peroxidation end products (ALEs). Ethanal,
propanal, hexanal, glyoxal, methylglyoxal, 4-hydroxy-2-hexenal, acrolein, formalde-
hyde, and acetaldehyde are considered ALEs. These are reactive carbonyl compounds
(RCCs) and sugar glycoxidation products (called advanced glycation end products)
that usually accumulate with aging and oxidative stress-related diseases such as
atherosclerosis, diabetes, or neurodegenerative diseases. RCCs induce the ‘carbonyl
stress’ characterized by the formation of adducts and cross-links on proteins, which
progressively leads to protein malfunction and damages in all tissues and patho-
logical consequences, including cytotoxicity, cell dysfunction, inflammation, and
apoptotic cell death. Further interaction with proteins by these ALEs can cause both
structural and functional changes of oxidized proteins. Specifically, 4-HNE can react
with lysine, histidine, or cysteine residues in protein to form adducts. The enzyme
immunoassay that is developed is for rapid detection and quantification of these
adducts. In these assays, the quantity of adduct in protein samples is determined by
comparing its absorbance with that of a known adduct – BSA standard curve.

LDL cholesterol, generally referred to as bad cholesterol, is even more dangerous

when it becomes oxidized. Oxidized LDL (OxLDL) is more reactive with surrounding
tissues and can collect within the inner lining of arteries. Macrophages, cholesterol,
and other lipids can accumulate at the site (atherosclerosis), ultimately forming a
plaque that can lead to heart attack, stroke, or death. LDL oxidation affects both the
lipid and protein components of LDL. Reactive aldehyde products formed during the
oxidation of PFAs, such as MDA and 4-HNE, are capable of attaching covalently to
the -amino groups of lysine residues of ApoB-100 to form MDA-Lys and HNE-Lys
adducts (MDA-LDL and HNE-LDL). Advanced glycosylation such as the formation of
CML-LDL and CEL-LDL is also involved in LDL oxidation. The enzyme immunoassay
is developed for the detection and quantitation of human OxLDL in plasma, serum
or other biological fluid samples. The kit, which is readily available, contains a copper
oxidized LDL standard.
Historically, the measurement of MDA by TBARS (thiobarbituric acid reactive sub-
stances) assay has been frequently used to determine the extent of LP. It is considered
nonspecific, and is generally poor when applied to biological samples. Recent assays
are based on the measurement of MDA or HNE-lysine adducts. These are likely to
be more applicable to biological samples, since adducts of these reactive aldehydes
are relatively stable. The discovery of the isoprostanes as lipid peroxidation products,
measured by GCMS or immunoassay, has opened a new avenue for indirect quan-
titation of lipid peroxidation in vivo.

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Review on the Protective Effects of

the Indigenous Indian Medicinal Plant,
Bael (Aegle marmelos Correa), in Gas-
trointestinal Disorders
M.S. Baliga, ... R. Jimmy, in Bioactive Food as Dietary Interventions for Liver and
Gastrointestinal Disease, 2013

10.2 Inhibition of LPx

LPx, which can occur through enzymatic or nonenzymatic reactions, is associated
with cellular damage and mutagenesis. Preclinical studies have shown that the
leaf extract decreases the LPx, conjugated dyne, and hydroperoxide levels in allox-
an-treated diabetic rats (Sabu and Kuttan, 2004). Additionally, the leaf and fruit are
also reported to decrease the CCl4- and alcohol-induced lipids in the liver (Khan
and Sultana, 2009; Singanan et al., 2007) and the radiation-induced LPx in the liver,
kidney, intestine, and spleen of mice (Jagetia et al., 2004a,b). All these results clearly
indicate that the inhibition of oxidant-induced LPx is a universal phenomenon and
a possible cause of the cytoprotective properties of Bael leaves.

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Antioxidants and Vitamins

Prabhakar Singh, ... Raj K. Keservani, in Sustained Energy for Enhanced Human
Functions and Activity, 2017
Oxidative Damage to Lipids
LPO was first studied in the 1930s in relation to the deterioration of food (Walling,
1955; Niki, 2000). Lipids regulate various important biological processes in the
cell because they are a major component of membranes and have several crucial
enzymes, and they represent a potent class of hormones. Because of increasing
evidence regarding the involvement of free radicals in biology (Halliwell and Gut-
teridge, 2006), LPO has received renewed attention from broader viewpoints in the
fields of chemistry, biochemistry, biology, nutrition, and medicine. Studies revealed
that the LPO proceeds by three distinct mechanisms: (1) free radical–mediated
oxidation; (2) free radical–independent, nonenzymatic oxidation; and (3) enzymatic
oxidation (Niki et al., 2005). Among these three, free radical–mediated LPO has been
found have plethoric health consequences. An increased concentration of LPO end
products is the most frequently quoted evidence for the involvement of free radicals.
The level of LPO products in the plasma of healthy human subjects is reported
to be below 1 μM and 0.1% of the parent lipids. An increased value of LPO and
LPO end products causes profound alterations in the structural organization and
functions of the cell membrane including decreased membrane fluidity, increased
membrane permeability, inactivation of membrane-bound enzymes, and the loss
of essential fatty acids (Ginkel and Sevanian, 1994). Increased LPO end products
have been shown to be mutagenic and carcinogenic (West and Marnett, 2006) and
have been implicated as the underlying mechanisms in cardiovascular diseases,
cancer, neurological disorders, and aging (Rizvi and Maurya, 2007b; Lee et al.,
2004). Substantial oxidative damage to membrane phospholipids occurs with aging
in both microsomes and plasma membranes, and is more prevalent in plasma
membranes in hepatocytes (Hayashi and Miyazawa, 1998). LPO induced modificat-
ions in proatherogenic and proinflammatory lipoprotein molecules that accelerated
the atherosclerosis process and associated vascular diseases. LPO products have
been found to induce various oxidative stress-mediated disorders including disease
in different models (Niki, 2009). Chemically reactive LPO products react directly
with thiol groups of Cys, histidine, and lysine residues in proteins and peptides
to form stable adducts and exert cytotoxic effects that contribute to the etiology
of various diseases, including neurodegenerative ones (Sultana et al., 2006). Reac-
tive unsaturated carbonyl compounds, lipid hydroperoxides, hydroxyl fatty acids,
hydroxylcholesterol, ketocholesterol, epoxycholesterol, and lysophosphatidylcholine
have been reported to reduce cellular activity and accelerate cell mortality (Piga et al.,
2007). Increased LPO and changes in the cholesterol/phospholipid molar ratio were
reported with altered Na+/K+-ATPase activity of erythrocytes of patients with cervical
cancer (Kolanjiappan et al., 2002). LPO products as signaling messengers have been
documented extensively (Zmijewski et al., 2005); however, to exert physiologically
important functions as a regulator of gene expression and a mediator of cellular sig-
naling, the formation of LPO products must be strictly controlled and programmed.

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Monitoring Vesicular Trafficking in

Cellular Responses to Stress - Part B
Jingbo Li, ... Daolin Tang, in Methods in Cell Biology, 2021

3.1 Overview
Lipid peroxidation is the core reaction of ferroptosis, which is caused by the attack of
oxidants on lipids. Due to the production of lipid peroxyl radicals, hydroperoxides,
and various oxidation products, uncontrolled lipid peroxidation leads to membrane
rupture and cell death. Commercial lipid peroxidation kits and probes can be used
to monitor the oxidative status of ferroptotic cells (Table 1). For example, malondi-
aldehyde (MDA) and 4-hydroxynonenal (4HNE) are the major products converted
from aldehydes during lipid peroxidation (Benedetti, Comporti, & Esterbauer, 1980;
Esterbauer, Cheeseman, Dianzani, Poli, & Slater, 1982; Jordan & Schenkman, 1982).
MDA interacts with thiobarbituric acid (TBA) and forms an MDA-TBA adduct, which
is sensitively detected and quantified by fluorescence colorimetry. Enzyme-linked
immunosorbent assay (ELISA) kit is used to quantify 4HNE adducts in samples.
Overall, these assay kits can easily quantify the by-products produced by lipid
peroxidation, which is important for evaluating autophagy-dependent ferroptosis.

Table 1. Commercial lipid peroxidation assay kits and probes.

Product name Target Sample Equipment Vender (Catalog

number)
Lipid peroxidation 4HNE-protein Purified protein, Microplate reader Abcam(ab238538)
(4HNE) assay kit adducts plasma, serum,
tissue lysate, and
cell lysate
HNE adduct com- 4HNE-protein Purified protein, Microplate reader Cell Biolabs-
petitive ELISA adducts plasma, serum, (STA-838)
tissue lysate, and
cell lysate
4-hydroxynonenal 4HNE-protein Serum, plas- Microplate reader Bio Vision(E4645)
(4HNE) ELISA kit adducts ma, tissue ho-
mogenate, and
other biological
fluids
Lipid peroxida- MDA Urine, plasma, cell Microplate reader Abcam(ab118970)
tion (MDA) as- culture extracts,
say kit (colorimet- and tissue extracts
ric/fluorometric)
MDA Microplate reader Abcam(ab233471)
Lipid peroxidation Urine, plasma, cell
(MDA) assay kit culture extract,
(colorimetric) and tissue extract
TBARS (lipid per- MDA Urine, plasma, Microplate reader Cell Biolabs-
oxidation) assay serum, lysate, (STA-330)
and tissue ho-
mogenate
Lipid peroxidation MDA Plasma, tissue, Microplate reader GENWAY-
(MDA) assay kit and cell extracts (GWB-AXR340)
Image-iT lipid per- BODIPY581/591 Living cells Floid cell imag- Thermo Fisher Sci-
oxidation kit C11 ing system, flow entific(C10445)
cytometer, high
content instru-
ment, and f-
luorescence micro-
scope
Liperfluo A Spy-LHP analog Living cells Flow cytometer Dojindo Molecu-
and fluorescence lar Technologies-
microscope (L248)
Click-iT lipid per- Alkyne-contain- Fixed cells Floid cell imag- Thermo Fisher Sci-
oxidation de- ing modified ing system, flow entific(C10446)
tection with proteins cytometer, high
linoleamide alkyne content instru-
(LAA) ment, and f-
luorescence micro-
scope

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Green Tea (−)-Epigallocatechin-3-Gal-

late and its Effects on Pancreatic Stel-
late Cells
Makoto Otsuki, Hiroshi Asaumi, in Tea in Health and Disease Prevention, 2013

Effects of EGCG on Lipid Peroxidation of the Cell Membrane

Lipid peroxidation of the cell membrane is examined by applying
diphenyl-1-pyrenylphosphine (DPPP) as it is a probe that is highly reactive against
hydroperoxides, and fluoresces when it reacts with lipid peroxidation (Takahashi
et al., 2001). PSCs cultured without any treatment demonstrated only low lev-
els of DPPP oxide fluorescence in the cell membranes after incubation for 24
h (Figure 108.4A), whereas high levels of fluorescence were observed in the cell
membrane when DPPP-labeled PSCs were cultured with ethanol (Figure 108.4B).
The ethanol-induced lipid peroxidation is sustained over the 24-h incubation. These
changes in fluorescence intensity are similarly observed in cell membranes of all
PSCs. The strong anti-oxidant EGCG prevents lipid peroxidation in cell membranes
and suppresses fluorescence in PSCs incubated with ethanol in a concentration-de-
pendent manner (Figure 108.4C, D).
Figure 108.4. Fluorescence Microscopic Images of PSCs Treated with DPPP = O-la-
belled Lipid Hydroperoxide in the Cell Membrane.PSCs were incubated for 24 h;
(A) control, (B) with 50 mM ethanol, (C) 50 mM ethanol plus 5 μM EGCG, and (D)
50 mM ethanol plus 25 μM EGCG.The PSCs without any treatment demonstrated
low levels of fluorescence derived from DPPP oxide in the cell membranes (A),
whereas high levels of fluorescence in the cell membranes were detected after
incubation with ethanol (B). EGCG suppressed ethanol-induced fluorescence in a
concentration-dependent manner (C, D). Original magnification 400 × objective.
(Modified from Asaumi et al., 2006, with permission.)

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Plant Secondary Metabolites With He-

patoprotective Efficacy
Ashutosh Gupta, Abhay K. Pandey, in Nutraceuticals and Natural Product Pharma-
ceuticals, 2019

3.4.2 Lipid Peroxidation and Redox Cycling

Lipid peroxidation is the most common factor associated with cell death due to
the overproduction of free radicals which is caused by alteration in the intracellular
pro-oxidant to antioxidant equilibrium in favor of pro-oxidants (Sharma et al., 2017).
Lipid peroxy radicals increases cell membrane permeability, membrane proteins in-
activation, decreases cell membrane fluidity, and loss of mitochondrial membranes
polarity. Metal ions contribute in redox cycling while cycling of oxidized and reduced
forms of a toxicant lead to the production of free radicals which suppress glutathione
activity by oxidizing critical protein sulfydryl groups participated in enzymatic or cel-
lular regulation or can induce lipid peroxidation. Intake of excessive ethanol triggers
free-radical production, lipid peroxidation, and glutathione reduction (Saukkonen
et al., 2006). Hydrocarbons linked with halogen, hydroperoxides, sodium vanadate,
iodoacetamide, cadmium, acrylonitrile, and chloroacetamide are also suggested to
exhibit hepatotoxicity because of lipid peroxidation.

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Transcriptional Factor Modulation by

Lipid Peroxidation-Derived Aldehydes
Ashish Saxena, ... Kota V. Ramana, in The Molecular Nutrition of Fats, 2019

12 Conclusions
Lipid peroxidation-derived aldehydes play a major role in human health and disease
as they act as important signaling intermediates in modulating cellular physiological
function via regulating various transcription factors (Fig. 32.6). Their ability to cova-
lently modify different proteins, adduct formation with amino acids, glutathione,
nucleic acid bases significantly alters the cellular homeostasis during pathological
and oxidative stress conditions. Apart from its ability to affect the transcriptional
activity of various transcription factors, HNE has also been reported to affect a
plethora of other mechanisms and proteins in cells affecting cellular functions.
Modification of proteins and DNA by LDAs such as acrolein, MDA, crotanaldehyde,
and HNE have been attributed to various pathological conditions. Unsaturated
aldehydes have the ability to form adducts with deoxyguanosine (dG) residues and
lead to the formation of LDA-dG. LDAs have the ability to react with all the four DNA
bases but with different specificities, which results in the formation of cancerous
mutations in cells (Huang et al., 2010). Many investigators have highlighted the im-
portance of DNA–HNE adducts as important biomarkers of oxidative stress during
carcinogenesis (Breitzig et al., 2016). Further, recent studies have highlighted the
role of hydroxyalkenals in neuropsychiatric disorders which are linked to their ability
to induce changes in blood–brain barrier and potentially altering their permeability
(Romano et al., 2017).
Figure 32.6. Transcription factors modulation by lipid peroxidation-derived alde-
hydes.Various transcription factors are directly or indirectly activated or repressed
by LDAs leading to either activation or repression of genes. The modulation of
transcription factors is mainly due to the ability of LDAs to covalently modify proteins
and form adducts with nucleic acids, glutathione, and amino acids leading to
alteration in their function.

Many of LDAs and their conjugates with proteins have been identified as potential
biomarkers for various disease conditions. Various antioxidants and plant-derived
polyphenols that alter cellular levels of LDAs could be developed for the therapeutic
interventions for various disease conditions. Further, targeting various enzymes
like aldo-keto reductases, GSTs, and carbonyl reductases, which metabolize lipid
peroxidation-derived aldehydes have shown significant therapeutic potential for
preventing complications associated with oxidative stress including cancer.

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Chlorogenic Acids From Sweet Potato

Taihua Mu, ... Cheng Wang, in Sweet Potato Processing Technology, 2017

3.1.4 Peroxy Radical-Scavenging Activities of Chlorogenic Acids

From Sweet Potatoes
Lipid peroxidation is a major cause of many pathological phenomena. Peroxy rad-
icals are the intermediate products of lipid peroxidation, which can attack lipids,
causing the lipid peroxidation chain reaction.

The oxygen radical absorbance capacity (ORAC) method was used to determine the
peroxy radical-scavenging activity of chlorogenic acids from sweet potato leaves,
and the results are shown in Fig. 7.12. The ORAC of chlorogenic acids from sweet
potato leaves increased as the sample concentrations rose. When the concentration
of chlorogenic acids from sweet potato leaves was 20 μg/mL, the activity of “Yuzi
No.7” was 55.78 μg Trolox equivalent (TE)/mL, which was significantly higher than
the other samples, and was 2.8 times that of Trolox. The activity of “Ximeng No.1”
was 43.72 μg TE/mL, which was 2.2 times that of Trolox.

Figure 7.12. Oxygen radical absorbance capacity of chlorogenic acids from sweet
potato leaves.Note: a−b Values labeled with a different letter in the same concentration
are significantly different (P < 0.05).

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In Vitro Investigation of the Molecular

Mechanisms of Hepatotoxicity
JOSÉ V. CASTELL, ... ROQUE BORT, in In Vitro Methods in Pharmaceutical Re-
search, 1997
 1 Drug-derived radicals and active oxygen species
Lipid peroxidation is a mechanism of cell injury by chemicals,58–60 where drug
radicals generated during P450 metabolism often act as initiators. For example, in
the course of halogenated hydrocarbon metabolism (C13C, C14C), carbon-centred
radicals61 capable of initiating lipid peroxidation and are formed by halogen abstrac-
tion by the haem group of cytochromes.62,63

Another classical example is the metabolism of compounds like paraquat and di-
quat which undergo one-electron redox cycling reactions, generating continuously
drug-derived radicals64,65 and superoxide anion which becomes dismuted to H2O
and H2O2. The latter, by the Fenton-Harber-Weiss reaction, can yield OH· radicals
which, in turn are able to promote new lipid radicals by H abstraction. Lipid rad-
icals can react with molecular oxygen to yield unstable hydroperoxides that suffer
homolytic breakdown, generating new radicals.

Cells use several strategies to protect themselves from uncontrolled lipid peroxi-
dation: (a) inactivation of active oxygen species; (b) trapping of eventually formed
radicals; (c) inhibition of the radical chain propagation; and (d) repair of damaged
lipids. Superoxide dismutase, catalase and glutathione peroxidase, together with
reduced glutathione (GSH) are the most efficient cellular agents against oxygen
species and radicals. Natural antioxidants, such as vitamin E present in biological
membranes, act by inhibiting the propagation step of lipid peroxidation.66,67 Lipid
hydroperoxides are substrates of glutathione peroxidase. Using 2 mol of GSH, this
enzyme reduces the –OOH group to an alcohol. However, for this enzyme to act
efficiently, a minimal level of reduced glutathione needs to be present in cells.

Lipid peroxidation can be the direct consequence of drug-derived radical formation,

or may appear concomitantly after previous GSH depletion as the result of the cell's
inability to protect itself from active oxygen species generated during cellular ox-
idative metabolism.33,68 Lipid peroxidation often precedes irreversible cell damage,
being an early cause of cell death.33,63,69

An indirect way to assess the role of lipid peroxidation in the mechanism of cell injury
is to evaluate the effects of the xenobiotic in the presence or absence of antioxidant
agents like N,N -diphenylphenylenediamine, vitamin E and promethazine.58,69–71
However, it is not always possible to draw conclusions about what occurs first in the
course of cell injury. Even in cases in where lipid peroxidation has been shown not to
be the critical step in causing cell death, there is evidence showing that peroxidation
can act synergistically with other damaging mechanisms to amplify liver injury.70

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