Analytical Biochemistry 524 (2017) 13e30

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Assessment of lipid peroxidation by measuring malondialdehyde

(MDA) and relatives in biological samples: Analytical and biological
challenges
Dimitrios Tsikas*
Centre of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Malondialdehyde (MDA), 4-hydroxy-nonenal (HNE) and the F2-isoprostane 15(S)-8-iso-prostaglandin F2a
Received 12 October 2016 (15(S)-8-iso-PGF2a) are the best investigated products of lipid peroxidation. MDA, HNE and 15(S)-8-iso-
Accepted 21 October 2016 PGF2a are produced from polyunsaturated fatty acids (PUFAs) both by chemical reactions and by re-
Available online 24 October 2016
actions catalyzed by enzymes. 15(S)-8-iso-PGF2a and other F2-isoprostanes are derived exclusively from
arachidonic acid (AA). The number of PUFAs that may contribute to MDA and HNE is much higher. MDA is
Keywords:
the prototype of the so called thiobarbituric acid reactive substances (TBARS). MDA, HNE and 15(S)-8-iso-
Biomarkers
PGF2a are the most frequently measured biomarkers of oxidative stress, namely of lipid peroxidation. In
Lipid peroxidation
Oxidative stress
many diseases, higher concentrations of MDA, HNE and 15(S)-8-iso-PGF2a are measured in biological
PUFAs samples as compared to health. Therefore, elevated oxidative stress is generally regarded as a patho-
ROS logical condition. Decreasing the concentration of biomarkers of oxidative stress by changing life style, by
nutritional intake of antioxidants or by means of drugs is generally believed to be beneficial to health.
Reliable assessment of oxidative stress by measuring MDA, HNE and 15(S)-8-iso-PGF2a in biological fluids
is highly challenging for two important reasons: Because of the duality of oxidative stress, i.e., its origin
from chemical and enzymatic reactions, and because of pre-analytical and analytical issues. This article
focuses on these key issues. It reviews reported analytical methods and their principles for the quanti-
tative measurement of MDA, HNE and 15(S)-8-iso-PGF2a in biological samples including plasma and
urine, and critically discusses their biological and biomedical outcome which is rarely crystal clear and
free of artefacts.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
Reactive oxygen species (ROS) such as the superoxide radical
anion (O2
e) and the peroxide non-radical dianion (O2 2 ) can be
produced both by means of enzymes and by non-enzymatic
chemical reduction of molecular oxygen (O2). ROS are highly
reactive and attack in their vicinity various classes of biomolecules
including proteins, DNA and lipids such as polyunsaturated fatty
acids (PUFAs) (Scheme 1). This phenomenon is generally known as
“oxidative stress” or “oxidant stress”. The PUFA arachidonic acid is
peroxidized to finally form malondialdehyde (MDA), 4-hydroxy-2-
nonenal (HNE) and other reaction products such as F2-isoprostanes
* Centre of Pharmacology and Toxicology, Hannover Medical School, Carl-
Neuberg-Strasse 1, D-30625, Hannover, Germany.
E-mail address: tsikas.dimitros@mh-hannover.de.
(Scheme 1). The particular reaction of ROS with lipids is generally
known as “lipid peroxidation”. MDA, HNE and F2-isoprostanes are
widely accepted biomarkers of oxidative stress, namely of lipid
peroxidation.
The number of scientific articles reporting on oxidative stress in
general and lipid peroxidation in particular steadily increases, with
the proportion of lipid peroxidation, when represented by MDA,
seeming to increase in favor of lipid peroxidation over the last two
decades (Fig. 1). Generally, oxidative stress is considered hazardous
to health and it is, therefore, not surprising that the number of
scientific articles dealing with oxidative stress is remarkably high.
Oxidative stress and lipid peroxidation are central to modern hu-
man life (Table 1). The research area of oxidative stress is appre-
ciably challenging for many different reasons. Perhaps the most
important issue is its inherent complexity. Scientists in this area of
research are convinced that oxidative stress is closely associated
with disease, aging and even with dead. Analysis of scientific

http://dx.doi.org/10.1016/j.ab.2016.10.021
0003-2697/© 2016 Elsevier Inc. All rights reserved.
14 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30

Abbreviations MS mass spectrometry

PAOD peripheral arterial occlusive disease
AA arachidonic acid PAR peak area ratio
BHT butylated hydroxytoluene PFB pentafluorobenzyl
CID collision-induced dissociation PFB-Br pentafluorobenzyl bromide
COX cyclooxygenase PG prostaglandin
DAN 2,4-diaminonaphthalene 15(S)-8-iso-PGF2a 15(S)-8-iso-prostaglandin F2a
DHA docosahexaenoic acid PUFAs polyunsaturated fatty acids
DNPH 2,4-dinitrophenylhydrazine ROS reactive oxygen species
ECNICI electron-capture negative-ion chemical ionization RNS reactive nitrogen species
EPA eicosapentaenoic acid RSD relative standard deviation
GC-MS gas chromatography-mass spectrometry SIM selected-ion monitoring
GC-MS/MS gas chromatography-tandem mass spectrometry SRM selected-reaction monitoring
12-HHT 12(S)-hydroxy-heptadecatrienoic acid TBA thiobarbituric acid
HNE 4-hydroxy-2-nonenal TBARS thiobarbituric acid-reactive substances
HPLC high-performance liquid chromatography TMS trimethylsilyl
LOD limit of detection Tx Thromboxane
LOQ limit of quantitation TxB2 thromboxane B2
MDA malon(di)aldehyde

Fig. 1. Articles found in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) using the

Scheme 1. Simplified schematic illustrating the attack of reactive oxygen species (ROS)
on proteins, DNA and polyunsaturated fatty acids (PUFAs), the latter being represen-
tatives of the class of lipid biomolecules. ROS peroxidize PUFAs to finally generate
malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE). The eicosanoid PUFA
arachidonic acid is peroxidized by ROS to form F2-isoprostanes such as 15(S)-8-iso-
prostaglandin F2a in addition to MDA and HNE. Lipid peroxidation may be enzymatic
and non-enzymatic in nature.
literature reports reveals that oxidative stress is in the focus of
scientists from many different disciplines, including analytical
chemists, physicians, pharmacologists and toxicologists, sport
medicine specialists, and nutritionists (Table 1).
The present article reviews and discusses the methodological
issues of MDA measurement in biological samples and attempts to
evaluate the significance of MDA as a biomarker of lipid peroxi-
dation in health, disease and lifestyle in modern times.
2. Chemistry and biology of malondialdehyde
At room temperature, malondialdehyde (MDA) or 1,3-
propanedial (OHCeCH2eCHO; C3H4O2; MW, 72.06; CAS 542-78-
search term (A) “oxidative stress” and (B) “malondialdehyde” AND “oxidative stress” in
the title and/or abstract of the articles from 1990 to 2015. (A:B) Ratio of the number of
articles found by (A) to that found by (B). Since about 1997, the proportion of papers
reporting on malondialdehyde in the oxidative stress areas of research increases
constantly over the years.
9; PubChem, 10964) is a solid compound (melting point, 72e74  C).
Authentic MDA is not commercially available. MDA is easily
released from commercially available precursors such as 1,1,3,3-
tetraethoxypropane by acid-catalyzed hydrolysis (e.g., 0.1 M HCl)
(Scheme 2A) [1]. MDA is soluble in water, methanol and ethanol,
moderately soluble in methylene chloride and insoluble in diethyl
ether [2]. In aqueous solution MDA exists in two forms with two
distinct UV maxima depending on the pH: the maximum absor-
bance wavelength is 245 nM (ε 12,800) for pH < 3 and 267 nm (ε
29,400) for pH > 7. MDA is a CH-acidic dicarbonyl compound, a
protonic acid in aqueous solution: with a pKa value of 4.46 [3,4],
MDA is as acidic as aliphatic carboxylic acids (Scheme 2B). Due to its
carbonylic functionalities, MDA is chemically reactive. It polymer-
izes easily and undergoes many reactions with nucleophilic centers
of various biomolecules including DNA and amino acids moieties in
 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
proteins. From an analytical perspective, the chemical properties of
MDA can be divided into two categories, i.e., according to 1) to the
CH-acidity of the methylene H atoms; and 2) the reactivity of its
two aldehyde groups towards nucleophiles. These features have
been utilized for the measurement of MDA in biological samples
and will be oulined below. For a detailed review and discussion of
the biochemistry and biochemistry of MDA and other aldehydes see
the pioneer paper by Esterbauer and colleagues [5].
Numerous in vitro and in vivo studies have been dedicated to
the biology of MDA in humans especially including its toxicological,
mutagenic and cancerogenic potential. Data on MDA were
reviewed in IARC, and MDA was classified in IARC Monographs
Supplement 7 (1987). In 1999, it has been reported that MDA is “not
classified as to its carcinogenicity to humans (Group 3)” [6]. The
chemistry and biology of DNA damage induced by MDA has been
reviewed by many groups over the last three decades. Marnett
concluded in his review that “MDA may contribute significantly to
cancer linked to lifestyle and dietary factors” [7]. The group by
Marnett reported in 2003 that MDA is mutagenic in human cells
[8]. However, it must be noted that in the study by Niedernhofer
and colleagues mutagenic effects were observed at MDA concen-
trations being up to 6 orders of magnitude than pathophysiological
MDA levels in biological fluids and tissues [8]. Like MDA, F2-iso-
prostanes such as 8-iso-prostaglandin F2a (8-iso-PGF2a) exert
measurable biological activity at concentrations far above patho-
physiological concentrations. Nevertheless, the lipid peroxidation
products F2-isoprostanes [9], MDA [8] and HNE are considered not
only biomarkers of oxidative stress but also biologically active
compounds of physiological and pathological relevance.
3. Origin of biological malondialdehyde
Bernheim et al. published one of the earliest reports on the re-
action of thiobarbituric acid (TBA) with oxidation products of lipids
[10]. This group used the TBA reagent to detect oxidation products
of unsaturated fatty acids [11]. Hamberg & Samuelsson investigated
the enzymatic oxidation of the PUFAs linoleic acid, eicosatrienoic
acid and eicosatetraenoic acid (i.e., arachidonic acid, AA) by vesic-
ular gland of sheep [12]. In addition to the identification of 12-
hydroxy-8,10-heptadecadienoic acid from 8,11,14-eicosatrienoic
acid by mass spectrometry (MS), Hamberg & Samuelsson identi-
fied MDA by means of the TBA reaction, as well as in the form of d-
N-2(pyrimidinyl)-L-ornithine after reacting MDA with L-arginine
under strong acidic conditions [12]. Hamberg & Samuelsson sug-
gested a common mechanism for the formation of MDA and 12-
hydroxy-8,10-heptadecadienoic acid from 8,11,14-eicosatrienoic
acid, which includes the formation of a cyclic peroxide [12]
(Scheme 3), analogous to the endoperoxide formed in the prosta-
glandin (PG) synthesis via the intermediate PGG2 [13]. Under
similar conditions and on a molar basis, the highest MDA amounts
were produced from 8,11,14-eicosatrienoic acid followed by
5,8,11,14-eicosatetraenoic acid (arachidonic acid) and 6,9,12-
octadecatrienoic acid (g-linolenic acid). The lowest MDA amounts
were found to be formed from 9,12,15-octadecatrienoic acid (lino-
leic acid) and 15-methyl-8,11,14-eicosatrienoic acid [12].
Induction of platelet aggregation by ADP, epinephrine, collagen
or thrombin produces small amounts of MDA, whereas arachidonic
acid-induced platelet aggregation is associated with the formation
of large amounts of MDA, which is inhibited by cyclooxygenase
(COX) inhibitors acetylsalicylic acid and indomethacin [14,15]. The
measurement of MDA in platelet-rich plasma was suggested as an
indicator of platelet prostaglandin synthesis from arachidonic acid
[14].
In platelet-rich plasma obtained from healthy subjects who
ingested a single dose of 600 mg aspirin the concentration of
15
thromboxane B2 (TxB2), the stable metabolite of thromboxane A2
(TxA2), decreased almost completely one and two days after
ingestion, whereas the concentration of MDA decreased by about
70% after two and three days [16]. Subsequently, the concentration
of TxB2 and MDA in platelet-rich plasma increased in parallel and
reached their baseline values (7 nmol MDA/109 platelets) on day 9
[16]. These observations indicated that MDA formation parallels
enzymatic synthesis of TxA2 in the platelets. The formation of MDA
and TxA2 in intact human platelets was attributed to TxA2 synthase
[17]. Human platelets microsomes were found to convert PGH2 to
TxB2, 12(S)-hydroxy-5,8,10-heptadecatrienoic acid and MDA (as
measured by the batch TBA assay) in approximately equimolar
amounts [18]. Partially purified thromboxane synthase from hu-
man platelets was found to convert PGG2 to 12-hydroperoxy-
5,8,10-heptadecatrienoic acid [19], the precursor of 12-hydroxy-
5,8,10-heptadecatrienoic acid, suggesting co-formation of MDA
which, however, had not been measured in the study by
Hammarstro €m [19].
The antioxidant glutathione (GSH) is oxidized to its thiyl free
radical by prostaglandin H synthase (PGHS) [20], commonly named
cyclooxygenase (COX). GSH is involved in the metabolism of pros-
taglandins and can inhibit PGHS activity as well [21,22]. In COX-1
isoform expressing human platelets, formation of TxA2 has been
reported to be accompanied by temporary formation in consider-
able amounts of oxidized glutathione, i.e., glutathione disulfide
(GSSG), MDA and 12-hydroxy-5,8,10-heptadecatrienoic acid
[16,23e25]. By using recombinant COX-1 and COX-2 we found that
these enzymes oxidize arachidonic acid to MDA, 12-hydroxy-
5,8,10-heptadecatrienoic acid and 15(S)-8-iso-prostaglandin F2a
(15(S)-8-iso-PGF2a) in addition to the classical prostaglandins PGE2,
PGD2, PGF2a and TxB2 [26]. We found that GSH promotes
concomitant COX-catalyzed conversion of arachidonic acid to MDA
and 15(S)-8-iso-PGF2a in a concentration-dependent, yet non-
linear fashion. The concentrations of MDA and 15(S)-8-iso-PGF2a
correlated very closely with each other both for COX-1 (r ¼ 0.97)
and COX-2 (r ¼ 0.98), with the concentration of MDA being 100 to
200 times higher than that of 15(S)-8-iso-PGF2a [26].
With respect to origin, mechanisms of formation, chemistry and
biology of MDA, HNE and 15(S)-8-iso-PGF2a is referred to recent
reviews [27,28].
3.1. Malondialdehyde and its relation to other lipid peroxidation
biomarkers
Biological MDA exists primarily in two forms, i.e., free or cova-
lently bound to/conjugated with proteins, nucleic acids, lipopro-
teins and certain amino acids. Circulating MDA and MDA adducted
to soluble amino acids are excreted in the urine. Urinary amino acid
adducts of MDA include N-epsilon-(2-propenal)lysine, N-a-acetyl-
(epsilon)-(2-propenal)lysine, N-(2-propenal) serine and N-(2-
propenal)ethanolamine [29e32]. Free non-adducted and non-
protein-bound MDA seems not to occur in human plasma [33].
MDA, HNE, 15(S)-8-iso-PGF2a and other F2-isoprostanes are
considered end-products of lipid peroxidation of PUFAs especially
including arachidonic acid [34e36]. Circulating MDA is one of the
most commonly and widely used oxidative stress biomarkers
[34e36]. Oxidative stress is generally considered a major contrib-
utor to numerous diseases such as cancer, diabetes, asthma,
atherosclerosis, and Alzheimer's and Parkinson's diseases [35,36]
(see also Table 1).
The biological formation and the physiological occurrence of 12-
hydroxy-5,8,10-heptadecatrienoic acid and its metabolite 12-oxo-
5,8,10-heptadecatrienoic acid have been identified and measured
by several groups [12,18,19,26,37,38]. Yet, quantitative data
regarding correlation between MDA and 12-hydroxy-5,8,10-
16 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30

Table 1
Summary of the results found by searching the PubMed databank (http://www.ncbi. Table 1 (continued )
nlm.nih.gov/pubmed) for the listed keywords and their combinations in the title and
in the abstract. Keyword in title and abstract Article number

Keyword in title and abstract Article number Platelets 316

Cerebrospinal fluid 76
Oxidative stress 161,014 Plasma 5360
Malondialdehyde and oxidative stress 11,779 (7.3% of total) Saliva 53
Analysis/assay/measurement 3259/1950/813 Serum 6745
TBA/TBARS 305/400 Urine 665
HPLC 618 Albumin 743
Mass spectrometry 276 Hemoglobin 473
GC-MS 90 Carbon monoxide 53
GC-MS-MS/GC-MS/MS 6/3 Carbon tetrachloride (CCl4) 357
LC-MS-MS/LC-MS/MS 34/26 Hydrogen sulfide 93
Child 27 Nitric oxide 2908
Men 479 Ozone 84
Women 724 Fatty acid
Pregnancy 290 Arachidonic acid 625
4-Hydroxy-2-nonenal 173 Eicosapentaenoic acid 49
Isoprostane 174 Linoleic acid 146
Peroxynitrite 173 Oleic acid 64
Prostaglandin 472 Cyclooxygenase 344
Thromboxane 210 Nitric oxide synthase 899
Signaling 667 Xanthine oxidase 530
Alzheimer 55
Cancer 800
COPD 85
Diabetes 1621 heptadecatrienoic acid are very rare. In plasma of 20 elderly sub-
Glucose 1864
jects (15 males, 5 females) suffering from peripheral arterial
Hypercapnia 9
Hyperoxia 85
occlusive disease (PAOD), we observed close correlations between
Hypertension 548 MDA and total (i.e., free and esterified to lipids) 15(S)-8-iso-PGF2a
Hypoxia 510 (r ¼ 0.562, P < 0.001), as well as between MDA and HNE (r ¼ 0.798,
Inflammation 2244 P < 0.0001) [26,39]. It is worth mentioning that the molar ratio of
Insulin 860
MDA:HNE:15(S)-8-iso-PGF2a in this population was about
Ischemia 2666
Multiple sclerosis 38 500:250:1, with the plasma MDA concentration ranging between
Parkinson 21 300 and 3000 nM [26,39].
Preeclampsia 101 Previous in vitro studies revealed that photo-irradiation of
Rheumatism 7
linolenic acid ((9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid) and
Stroke 251
Transplantation 338
linoleic acid ((9Z,12Z)-octadeca-9,12-dienoic acid), but not of oleic
Aging 577 acid ((9Z)-octadeca-9-enoic acid) or stearic acid (octadecanoic
Chocolate 6 acid), produced 12 mmol MDA/3.6 mmol (0.33%) and 1.5 mmol MDA/
Drugs/Chemicals 680 3.6 mmol (0.04%), respectively, as measured by gas chromatog-
N-Acetylcysteine 341
raphy (GC) with nitrogen/phosphorus detection (NPD) after
Acetylsalicylic acid/Aspirin 40/133
ACE inhibitor 28 derivatization with methyl hydrazine [40]. By using the same GC-
Acetaminophen/Paracetamol 125/45 NPD method, this group found that oxidation of arachidonic acid
Allopurinol 126 with Fe2þ/H2O2 resulted in formation of MDA (25 nmol/3.3 mmol,
Carvedilol/Metoprolol/Nebivolol 44/17/16 0.8%) and HNE (9 nmol/3.3 mmol, 0.3%). Fe2þ/H2O2einduced
Nitroglycerin 9
Statin 66
oxidation of linoleic acid resulted in formation of MDA (53 nmol/
Exercise, physical 55 3.6 mmol, 1.5%) and HNE (13 nmol/3.6 mmol, 0.4%). Unlike arach-
Diving 10 idonic acid, the oxidation of linoleic acid (free acid) produced more
Marathon 8 MDA and HNE than the oxidation of their ethyl esters [41]. These
Fish 409
data suggest that, on a molar basis, Fe2þ/H2O2einduced oxidation
Fish oil 93
Lifestyle 46 of PUFAs will generate more MDA than HNE.
Meat 153 Based on the currently available in vitro and in vivo data on
Nutrition 134 formation and concentration in biological samples of MDA, HNE
Smoking 331 and 15(S)-8-iso-PGF2a we proposed mechanisms that may explain
Supplementation 1687
Wine 51
both their formation from peroxidation of arachidonic acid and
Blood 6399 their quantitative relationship (Scheme 4). These mechanisms are
Brain 2192 supported by findings from some groups (e.g., [12,13]), but differ in
Heart 1705 part from those reported/discussed by others (e.g., [27,28]).
Kidney 2142
Recently, we observed both in healthy elderly subjects and in
Liver 5009
Lung 1444 PAOD patients a close correlation between urinary MDA and uri-
Neuron 156 nary nitrite [42]. This may be of particular interest because MDA
Anti-coagulation 6 and nitrite are considered to reflect different types of oxidative
Coagulation 65 stress, i.e., lipid peroxidation and nitrosative stress, respectively.
Erythrocytes 754
Lymphocytes 290
The physiological occurrence of nitrated fatty acids such as nitro-
Macrophages 396 oleic acid [43], suggests that lipid peroxidation may be closer
Mitochondria 735 related to nitrosative stress than assumed thus far. The common
 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30 17

Scheme 2. (A) Hydrochloric acid-catalyzed hydrolysis of the commercially available 1,1,3,3-tetraethoxypropane releases 1,3-propanal, i.e., malondialdehyde (MDA). (B) MDA is a CH-
acidic compound (pKa, 4.46). In aqueous solutions with a physiological plasma pH value of 7.4, MDA ionizes entirely to produce the carbanion which is in equilibrium with its
isomeric enolate anion.

Scheme 3. Mode of enzymatic formation of malondialdehyde (MDA) and 12-hydroxy-8,10-heptadecadienoic acid from 8,11,14-eicosatrienoic acid (left panel), and of MDA and 12-
hydroxy-5,8,10-heptadecatrienoic acid from 5,8,10,12-eicosatetraenoic acid (right panel) as proposed by Hamberg & Samuelsson [12,13], respectively.

link between MDA and nitrite could be myeloperoxidase [42],
which oxidizes nitrite by hypochlorous acid (HOCl) to the nitrating
and oxidizing intermediate nitryl chloride (NO2Cl). Yet, this issue
warrants further investigations.
4. Analytical methods for MDA
4.1. General aspects
Numerous analytical methods have been reported over the
years for the measurement of MDA, HNE and 15(S)-8-iso-PGF2a in
biological samples. Reported analytical methods for biological MDA
have been thoroughly reviewed and discussed. For a recent review
see Ref. [44]. The present review will focus on analytical methods
for MDA, but will also consider analytical methods reported for
HNE for the assessment of lipid peroxidation.
In general, analytical methods take advance of the physico-
chemical properties of the analytes. As native MDA absorbs light in
aqueous solutions of pH < 3 at 245 nm or at 267 nm of pH < 7, this
property has been utilized to measure native MDA by spectro-
photometry [1] or in combination with high-performance liquid
chromatography (HPLC) [45]. However, HPLC with ultraviolet
absorbance detection with preceding protein precipitation with
acetonitrile seems not to allow quantification of MDA in plasma of
healthy human at physiological concentrations in the range
0e0.5 mM [45].
18 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
Scheme 4. Simplified proposed mechanisms for the formation of MDA (1,3-propanedial), 12-hydroxy-5,8,10-heptadecatrienoic acid, 4-hydroxy-2-nonenal, and 15(S)-8-iso-PGF2a
from COX-catalyzed peroxidation of arachidonic acid. For simplicity intermediates and the involvement of GSH are not shown. For more details see Refs. [26,39].
The limit of detection (LOD) for many analytes can be greatly
decreased by analytical derivatization, i.e., after chemical reaction
of the analytes with suitable derivatization reagents after careful
consideration of the physicochemical properties of the analytes and
the availability of analytical facilities (e.g., HPLC, GC-MS). The most
conventional approach involves spectrophotometric or fluorimetric
methods, which are referred to as “batch” assays in this article as
they do not involve any chromatographic or electrophoretic sepa-
ration. The most widely utilized chemical property of MDA and
other aldehydes including HNE in analytical chemistry is their
aldehyde functional groups which are reactive towards chemicals
with nucleophilic groups (Scheme 5A, B). The second characteristic
chemical property of MDA is the considerable CeH acidity of the
methylene H atoms in aqueous solution [3,4] (Scheme 2B). The
reactivity of the carbonylic groups of MDA has been widely used for
the measurement of MDA in biological samples (Scheme 5A, B).
Recently, we have utilized the CH-acidity, i.e., nucleophilicity, of
MDA for its derivatization with the electrophilic derivatization re-
agent pentafluorobenzyl bromide (PFBeBr) in aqueous acetone
(Scheme 5C). The pentafluorobenzyl (PFB) derivative of MDA, i.e.,
(PFB)2MDA, is best suited for the quantitative GC-MS analysis of
native, non-conjugated MDA in biological samples including hu-
man plasma and urine [26,33,46].
4.2. Batch thiobarbituric acid-reactive substances assays
Chemical analysis of MDA started with its measurement as a
component of the so-called thiobarbituric acid-reactive substances
(TBARS) for the assessment of lipid peroxidation [47] by spectro-
photometry [48] or fluorimetry [49]. Under acidic conditions (e.g.,
glacial acetic acid or sulfuric acid) and elevated temperatures (e.g.,
95  C) for extended reaction time (e.g., 60 min), one molecule of
MDA reacts with two molecules of TBA to form a red-coloured,
strongly visible light-absorbing (lmax, 532 nm) and fluorescent
(lex, 515 nm; lem, 553 nm) derivative or TBA-MDA condensation
adduct (due to loss of the two O atoms of MDA as water). This
derivatives is usually abbreviated as TBA-MDA or MDA-(TBA)2
(Scheme 6). This is a Michael-like addition reaction in which the
nucleophilic centre is the aromatic ring of TBA. Under the deriva-
tization conditions the potentially stronger nucleophilic SH and OH
groups of TBA seem not to react with the carbonylic groups of MDA.
The batch spectrophotometric and spectrofluorometric TBA-
based methods are the most commonly used assays to measure
lipid peroxidation. However, they lack specificity because many
chemically reactive carbonyl groups-containing compounds from
different classes of substances, including oxidized polyunsaturated
fatty acids and carbohydrates, from endogenous sources and foods
present in body fluids can react with TBA and interfere with MDA
analysis. As an example, HPLC analysis of TBA-treated extracts of
oxidized methyl esters of linoleic acid and arachidonic acid
revealed formation not only of the TBA-MDA derivative, but also
several not identified TBA derivatives [50] (see also Ref. [49]. The
specificity of the batch TBARS assays can be considerably improved
by separating the MDA-(TBA)2 adduct from TBA adducts of other
TBARS substances, for instance by HPLC [51e53].
4.3. Chromatographic and mass spectrometric assays for MDA
Besides TBA many other derivatization reagents have been used
for the analysis of MDA by HPLC with visible absorbance or fluo-
rescence detection, GC-MS, GC-MS/MS or LC-MS/MS. The majority
of the methods uses derivatization reagents that react with the
carbonyl groups of MDA. Used derivatization reagents include 2,4-
dinitrophenyl hydrazine (DNPH) [54e56] and 2,4-
diaminonaphthalene (DAN) [57] in HPLC, and methyl hydrazine
[58,59], phenyl hydrazine [60], DNPH [61], 2-hydrazino-benzthia-
zol [62], and pentafluorophenyl hydrazine [63] in GC and GC-MS,
and 3-nitrophenyl hydrazine in the LC-MS/MS measurement of
MDA in human plasma [64].
HPLC, LC-MS/MS and GC-MS methods have been demonstrated
 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30 19

Scheme 5. Simplified schematic of three different reactions that are utilized in MDA analysis in biological samples. (A) Classical thiobarbituric acid (TBA) assay: one MDA molecule
reacts with two TBA molecules in the heat to form the MDA-(TBA)2 derivative. (B) Reaction of two 2,3-dinitrophenyl hydrazine (DNPH) molecules with the carbonylic groups of MDA
to form the hydrazone derivative. (C) At neutral pH in aqueous acetone, one MDA molecule reacts with two pentafluorobenzyl (PFB) bromide molecules to form the MDA-(PFB)2
derivative.

to be specific and more sensitive than the batch TBARS assays, and
moreover, to be useful both for free and adducted MDA [60,62].
More recently, we introduced pentafluorobenzyl bromide (PFBeBr)
[33], which alkylates MDA on its central C atom and lets both
carbonylic groups intact (Scheme 5C). The MDA-(PFB)2 derivative is
best useful for GC-MS and GC-MS/MS analysis of MDA from bio-
logical samples including human plasma and urine [26,33,46].
Although not directly compared, measurement of MDA as TBA
derivative with HPLC with fluorescent detection seems to provide
remarkably different results than HPLC analysis of MDA derivatized
with DAN and DNPH [65]. It is worth mentioning that derivatiza-
tion of MDA with TBA requires very high temperatures compared to
DAN, DNPH and other derivatization reagents that react with the
aldehyde moieties of MDA, as well as compared with PFB-Br which,
moreover, reacts with MDA at neutral pH or at the respective urine-
pH values, i.e., without any pH adjustment [33]. It should be point
out that DAN is not specific for MDA. Under the acidic conditions
required for the derivatization MDA other aldehydes [66] and ni-
trite [67] react with DAN to form derivatives that need to be
chromatographically separated for reliable measurement of MDA.
4.4. Measurement of free and bound MDA in protein-rich samples
Endogenous and exogenous MDA are extensively metabolized to
many metabolites including CO2, acetate and adducts to various
amino acids such as lysine and serine, as well as to ethanolamine
and guanine [68,69]. N-ε-(2-Propenal)lysine, N-a-acetyl-ε-(2-
propenal)lysine, N-(2-propenal) serine and N-(2-propenal)etha-
nolamine have been identified in urine [29e32]. The occurrence of
these MDA adducts in plasma has been only little investigated.
20 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
Scheme 6. Reaction of thiobarbituric acid (TBA) with MDA, representing a TBA-reactive substance, to form a highly delocalized system, a visible light-absorbing and strongly
fluorescent MDA-(TBA)2 derivative.
We have investigated the occurrence of MDA in its free form,
non-covalently bound to plasma proteins by ultrafiltration of hu-
man plasma. Despite the use of a highly sensitive GC-MS method
we did not detect free MDA in plasma of healthy humans [33],
suggesting that MDA is entirely non-covalently bound to plasma
proteins.
Another commonly used approach to generate free MDA is
protein precipitation by acids, for instance by perchloric acid, and
by analyzing the supernatant for MDA. HPLC analysis of MDA as
DNPH derivative provided a plasma concentration of free MDA
close to the limit of detection of the method which was 25 nM [70].
For the determination of total MDA, free þ protein bound, the
plasma sample was first treated with alkali (e.g., 30 min at 60  C),
then proteins were precipitated, and MDA was analyzed by HPLC as
DNPH derivative [70]. By this method, total MDA concentration was
measured to be 2.2 ± 0.3 mM in plasma healthy humans at baseline,
suggesting that the majority of circulating MDA is bound to plasma
proteins. Similar results for free and bound MDA in human plasma
were obtained by using a similar alkalinization/acidification pro-
cedure and by analyzing MDA as MDA-TBA adduct by HPLC [71].
Both in plasma and in serum of healthy subjects, free MDA was
measured to be 43 nM and 42 nM, while in plasma of cancer pa-
tients and hemodialyzed patients average free MDA was found to
be elevated, i.e., 270 and 214 nM, respectively [71]. However, it is
unclear whether alkalinization/acidification releases MDA bound
non-covalently to proteins or it hydrolyzes MDA adducted/cova-
lently bound to certain amino acid moieties of plasma proteins. It
was assumed that about 83% of MDA is bound to amino groups of
plasma and serum proteins [71]. By means of a similar alkaliniza-
tion/acidification procedure, use of a stable-isotope dilution GC-MS
method and by analyzing MDA as phenyl hydrazine derivative
prepared under mild conditions (30 min, 25  C, pH 4) mean free
and total MDA concentrations were measured to be 140 nM and
1300 nM in fresh human plasma [60].
The issues described above for MDA also apply to HNE. For a
detailed discussion of the chemistry, analysis and relevancy of HNE
in the assessment of lipid peroxidation is referred to a recent re-
view [72].
4.5. In vivo models of oxidative stress and lipid peroxidation
Poisoning of animals with paracetamol (acetaminophen) or
carbon tetrachloride (CCl4) are the most widely used in vivo models
of oxidative stress/lipid peroxidation. But, these models suffer from
numerous shortcomings [73e77]. These models are highly invasive,
poorly reproducible, and lack satisfactorily reproducible pharma-
cokinetics. Moreover, induced oxidative stress is commonly very
high, secondary to the poisoning and the fulminant failure of vital
organs, notably of the liver. Also, these models are acute and not
familiar with the oxidative stress/lipid peroxidation that occurs
chronically in common everyday-life. These conclusions can be
drawn from comprehensive, multilaboratory, nationwide valida-
tion studies which searched for non-invasive biomarkers of
oxidative stress including lipid peroxidation by measuring oxida-
tion products of lipids, proteins, and DNA in blood, plasma, and
urine of CCl4-poisoned rats [73e77].
As an example, plasma concentrations of MDA and F2-iso-
prostanes and urinary concentrations of F2-isoprostanes increased
upon CCl4-administration (120 and 1200 mg/kg i.p.). The authors of
the study concluded that measurements of MDA and F2-iso-
prostanes in plasma and urine are potential candidates for general
biomarkers of oxidative stress [73]. By using this model, two COX
inhibitors (indomethacin and meclofenamic) affected the concen-
trations of MDA and F2-isoprostanes. Nevertheless, the authors
concluded that the results of the study provided evidence that MDA
and F2-isoprostanes primarily constitute markers of free radical-
induced oxidative stress [74]. Negative effects were also found in
rat models based on ozone (2 and 5 ppm) exposure [75,76], and of
endogenous plasma antioxidants upon endotoxin-treatment (LPS,
2.5 and 5 mg/kg i.v.) [77].
 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
Recently, we proposed a human model of oxidative stress and
demonstrated its principle utility in a proof-of-concept study [78].
This model is based on the oral ingestion of a single pharmaco-
logical dose of a 500-mg paracetamol (acetaminophen) tablet by
adult subjects and on measuring in plasma and urine di-
paracetamol and 3-nitro-paracetamol in addition to common
endogenous biomarkers of oxidative stress/lipid peroxidation
including MDA [33,78]. Potential advantages of the human para-
cetamol model of oxidative stress are the safety of paracetamol in
adults and children at the therapeutical single oral dose of about
10 mg/kg body weight and its well-defined and highly reproducible
pharmacokinetics in terms of bioavailability, maximum plasma
concentration, time to reach maximum plasma concentration, and
elimination half-life: these pharmacokinetic parameters vary by
only about 15% between individuals [79]. Another potential
advantage of this model is that paracetamol, at the dose of 10 mg/
kg, does not affect TxA2, MDA and 15(S)-8-iso-PGF2a formation [78],
and does not induce oxidative stress. This model remains to be
tested in vivo in healthy and diseased humans.
4.6. Assessment of lipid peroxidation by measuring MDA in
biological samples
Many pre-analytical factors may compromise the analytical
outcome of the assessment of lipid peroxidation by measuring MDA
in lipid-rich biological fluids such as plasma or serum and tissues
including cells. Main well-recognized pre-analytical pitfalls include
sampling, sample storage, and artefactual formation. Knowledge of
these potential sources of analytical uncertainty in the measure-
ment of MDA is important, and inclusion of proper measures in
study and analytical protocols are essential to minimize adverse
effects of pre-analytical issues on MDA measurements.
With regard to blood sampling two major factors are important:
anti-coagulation and hemolysis. The effects of these factors on the
measurement of circulating MDA have been investigated by many
groups. The effects of anti-coagulation on plasma MDA are con-
tradictory, probably depending upon the analytical method itself
[33,39]. Hemolysis, even if not visible, may considerably contribute
to artificial formation of MDA (discussed in Ref. [46]). The reasons
are likely to include ex vivo peroxidation of free and esterified
PUFAs by: 1) oxyhemoglobin and other hemoglobin species; and 2)
activating platelets to peroxidize arachidonic dependent on and
independent of platelet COX-1. Possible strategies to minimize
ex vivo coagulation/anti-coagulation-dependent formation of MDA
are the use of COX inhibitors such as acetylsalicylic acid or indo-
methacin to block its enzymatic formation [14,15,80] and the use of
radical scavengers such as butylated hydroxytoluene (BHT). The
utility of BHT is outlined below. In a direct comparison, we found
considerable differences for MDA concentration determined in
freshly prepared serum, in EDTA plasma and in heparinized plasma
samples from human blood. The lowest MDA levels were measured
is serum samples [33]. Interestingly, differences regarding
coagulation/anti-coagulation were much less pronounced in sam-
ples that have been stored at 80  C for about three years and
which had considerably higher MDA concentrations [39] than
immediately analyzed plasma and serum samples [33].
Storage of lipid-rich samples for subsequent MDA analysis is a
serious pre-analytical concern. We found that sample storage and
time of sample storage until sample work up and MDA analysis are
particularly serious factors even in placebo-controlled long-term
clinical studies [33,39]. This arises the question of the most proper
time of sample analysis for MDA and other lipid peroxidation bio-
markers such as HNE and 15(S)-8-iso-PGF2a. Should plasma/serum
samples be analyzed after collection of the baseline samples, or
should MDA be analyzed at the end of the study after completion of
21
the whole study? Because of the placebo-controlled studies and in
order to ensure very similar conditions for sample work up and
analysis, we decided in two previous studies to analyze the plasma
samples after completion of the clinical studies in which patients
suffering from cardiovascular diseases {PAOD or coronary arterial
disease (CAD)} ingested daily for 3 or 6 months 10 g L-arginine in
the verum group or 10 g mannitol in the placebo group, respec-
tively. We observed in both study groups considerably lower MDA,
HNE and 15(S)-8-iso-PGF2a concentrations in the plasma samples
after 3 or 6 months than in the samples collected at the beginning
[33,39]. In the urine samples collected in parallel in these studies no
such differences were observed for MDA, HNE and 15(S)-8-iso-
PGF2a in both groups, suggesting that storage time is not a concern
for urinary biomarkers of oxidative stress.
BHT is widely used as an anti-oxidant in vitro studies on
oxidative stress including lipid peroxidation, as well as a free
radical scavenger to avoid/minimize artificial ex vivo formation of
MDA, HNE, F2-isoprostanes and other oxidative stress biomarkers
in biological samples. However, systematic studies on the effects of
BHT on the concentration in plasma, serum and urine of MDA, HNE
and F2-isoprostanes are rare. Jentzsch and colleagues investigated
the effects of oxygen and BHT on the concentration of MDA in
human plasma by using a modified batch TBA assay in which n-
butanol extracts were used for spectrophotometrical analysis [81].
Molecular oxygen physiologically present in plasma was found to
result in about 30% higher MDA (i.e., TBARS) concentrations than in
its absence (i.e., plasma degassed by argon) both in healthy and in
diabetes mellitus subjects [81]. By this TBA assay, MDA concen-
trations were found not to differ between healthy and diabetic
humans. In that study, plasma deoxygenation was found to be more
effective than the use of BHT at concentrations below 2.5 mM [81].
This study has clearly demonstrated that the time of plasma storage
at 20  C had a much greater effect on MDA concentrations that the
use of BHT. By approximation, plasma MDA concentrations were
measured to be 0.4 mM after 2 weeks, 0.9 mM after 2 months, and
higher than 10 mM after 1 year of storage [81]. A remarkable effect
of storage time on MDA concentration has been observed even in a
narrow time window of a few days [82].
Considering the pioneer work by D.M. Lee on the MDA forma-
tion in stored plasma [83] and other reported studies on the effects
of storage time and externally added anti-oxidants (here only a few
were considered [33,39,81,82]) on MDA concentration in plasma
and serum, the most useful strategy in the assessment of lipid
peroxidation by measuring MDA in plasma or serum seems to be
their immediate analysis following blood sampling. Given the low
lipid content of urine, artefactual formation of MDA in collected
urine samples seems not to be relevant [33,39]. Yet, preferably
identical experimental conditions need to be used, as even the pH
value of the TBARS reaction mixture seems to greatly affect MDA
concentration. Thus, decreasing the reaction mixture pH from 2.4 to
1.2 doubled the MDA concentration in huma serum [82].
4.7. Evaluation of MDA as a measure of lipid peroxidation in health,
disease and lifestyle
As discussed in the section above and in other parts of this
article, reliable quantitative measurement of MDA especially in
plasma and serum samples is challenging. A plethora of data on
MDA are found in the literature generated in vitro, in animal and in
nutritional and clinical studies investigating the effects of drugs,
anti-oxidants, and lifestyle including diet, smoking and physical
exercise. The use of different analytical methods and even the use
of the same analytical method hinders a dependable analysis of
reported studies and a reliable evaluation of MDA as a measure of
lipid peroxidation and its importance to human health. In many
22 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
assays including the TBA assay, it is actually uncertain whether the
same MDA species are detected, for instance authentic MDA, MDA
released from MDA-containing species and/or other TBARS. In
addition to purely analytical issues in terms of specificity, there are
pre-analytical „lipid peroxidation intrinsing“ factors which have
only rarely been considered appropriately. Thus, blood sampling,
coagulation, type of anticoagulation, preservation (e.g., deoxygen-
ation, use of BHT or other anti-oxidants), conditions and time
storage of protein-rich biological samples have been handled very
differently among scientific groups. These issues make difficult to
define reference values and intervalls for MDA in certain biological
samples such as plasma, serum and urine of humans. These issues
and the use of pathologically irrelevant, too high MDA concentra-
tions in vitro studies (e.g. Ref. [8]) are likely to have contributed to
the general belief that oxidative stress and lipid peroxidation are
involved in numerous human diseases [35,36] (Table 1).
A serious attempt to define reference intervals and to study
effects of lifestyle factors on plasma MDA was made by Nielsen and
colleagues [84]. The main results of the study by Nielsen et al. [84]
are worth mentioning and will be briefly summarized as follows.
The concentration of MDA was determined in plasma from
venous, with EDTA (about 5 mM) anticoagulated blood donated by
randomly selected 107 men and 106 women, aged 20e79 years,
representative for the general Da €nish population. Blood samples
were collected within two months. Blood samples were immedi-
ately centrifuged and 500-mL plasma aliquots were immediately
frozen to 80  C until analysis. Plasma MDA was measured by HPLC
with absorbance detection at 532 nm after appropriate method
validation. Unfortunately, the age of the plasma samples at the time
of analysis had not been reported. Nielsen et al. found a within-
subject (n ¼ 6) variation ranging between about 6 and 30% and
concluded that plasma MDA is unlikely to be useful as a biomarker
for the degree of lipid peroxidation on an individual basis but rather
on a group basis [84]. The plasma MDA concentration was found
not to change with the subjects age, whereas men were found to
have higher plasma MDA concentrations than women (0.41e1.29 vs
0.33e1.22 mM, P ¼ 0.003) [84]. Smokers (n ¼ 92) were found to have
higher mean plasma MDA concentrations than non-smokers
(n ¼ 122) (0.66 vs. 0.60 mM, P ¼ 0.05), whereas weekly alcohol
consumption correlated only very weekly with plasma MDA con-
centration (r ¼ 0.153, P ¼ 0.03). The effect of smoking on plasma
MDA concentration is contradictory in the literature. Smoking has
been reported to elevate (e.g. [85e87], and to not change (e.g.,
[88e90]) plasma MDA concentration.
Nielsen et al. reported that the MDA concentration in blood of 13
subjects was different in serum (1.16 mM), citrated plasma
(0.89 mM), heparinized plasma (1.27 mM), and EDTA plasma
(0.26 mM) [84]. These observations contradict our findings on MDA
serum (0.42 mM), heparinized plasma (0.59 mM) and EDTA plasma
(2.76 mM) [33] samples. Nielsen et al. argued that EDTA acts as an
anti-oxidant and prevents ex vivo formation of MDA, while the
conditions of serum generation favor MDA formation due to
platelet activation [84]. These are reasoned explanations but are not
supported by our observations. Thus, one may conclude that
coagulation/anti-coagulation may differently influence the
outcome of different methods of MDA analysis, e.g., MDA analysis
as MDA-(TBA)2 by HPLC [84] and MDA analysis as MDA-(PFB)2 by
GC-MS/MS [33].
It is believed that circadian rhythmicity is associated with
oxidative stress and their mutual impact may have implications for
human health and disease. In their review, Wilking and colleagues
concluded that „We believe that for a more efficacious manage-
ment of diseases that have both circadian rhythm and oxidative
stress components in their pathogenesis, targeting both systems in
tandem would be far more successful” [91]. The incidence of
cardiovascular events is maximal in the morning, with platelets
being considered important contributors. In patients with CAD,
platelet-derived TxA2, measured as its major metabolite 11-
dehydro-TxB2 in urine, was found to rise in the night between
0 and 8 a.m., while the maximum 11-dehydro-TxB2 excretion in
healthy humans occurs at 8e12 a.m. and is about two times lower
compared to CAD patients [92]. Circadian rhythmicity of TxA2
synthesis in healthy subjects has been reported earlier by others
[93,94]. As platelets are a major contributor to TxA2 and MDA, a
circadian rhythmicity for circulating and excretory MDA is
expectable. Yet, the circadian rhythmicity of circulating and urinary
biomarkers of oxidative stress/lipid peroxidation including MDA is
controversial in the literature. In three healthy young non-smoking
men, we did not observe convincing evidence of a circadian change
of the plasma MDA concentration when determined in immedi-
ately analyzed heparinized plasma samples by GC-MS/MS [33]
(Fig. 2). No circadian rhythm for MDA was found by means of a
batch TBARS assay in healthy subjects [86]. In normal mice, plasma
MDA concentration (measured by a batch TBARS assay) was found
to obey circadian rhythmicity [95]. In urine of healthy and diabetic
subjects, MDA was found to obey circadian variation, with the
changes being higher in diabetes [96].
There is indication that fatty acid composition in brain samples
from Alzheimer disease patients is altered compared to that of age-
matched controls and that Nε-(malondialdehyde)lysine targets
important brain cortex proteins to a higher extent than in controls
[97]. However, on the basis of circulating/excretory MDA there is no
convincingly evidence that lipid peroxidation plays an important
role in Alzheimer disease pathogenesis.
Mitochondrial impairment and oxidative stress are widely
considered to be central to the Parkinson disease. In a recent article,
Sanders and Greenamyre reviewed and discussed the oxidative
damage to macromolecules in the human Parkinson disease and in
the animal rotenone model [98]. The authors found that data on
plasma/serum MDA concentrations in Parkinson disease patients
are contradictory. Rotenone is an inhibitor of complex I. In vitro and
animal rotenone models seem to be consistent with regard to
elevated lipid peroxidation. Yet, in some studies it was not clear
which lipid peroxidation products have been measured with the
Fig. 2. Plasma malondialdehyde (MDA) concentrations measured in three healthy
non-smoking males (A, B, C). The first blood samples were obtained between 12:00
and 14:00, the last blood samples were collected after 24e28 h. The dotted line in-
dicates the mean MDA concentration considering all data. The numbers on the top of
the graph are the means ± standard deviation (relative standard deviation, %). MDA
was measured in the plasma samples a few days after sample storage at 80  C by GC-
MS/MS as described [33]. The main result of this experiment was reported earlier yet
without providing the individual concentrations [33].
 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
assays used. As to the effects of anti-oxidants in the Parkinson
disease, Sanders and Greenamyre concluded that the applied
strategies have had limited success in clinical trials [98]. For a
critical discussion on the outcome of anti-oxidant strategies in
health and disease see the review article by Giustarini et al. [36] and
Aldini et al. [99].
In diabetes mellitus, both in type 1 (T1DM) and type 2 (T2DM),
oxidative stress is generally accepted to play a fundament role.
Unlike in other diseases such as Alzheimer's and Parkinson disease,
the b-cell is considered extremely succeptible to oxidative stress,
mainly because of their weak antioxidative defence mechanisms
[100]. Yet, reported data of circulating an excretory MDA concen-
trations in T1DM and T2DM are contradictory as well. Given the
plethora of articles reporting on oxidative stress/lipid peroxidation
in diabetes mellitus (Table 1), here only a small number of articles is
discussed. Papers were selected in which MDA and/or F2-iso-
prostanes were measured in plasma/serum and urine of diabetic
patients with satisfactorily reported analytical methods. Jentzsch
et al. did not find different MDA concentrations in plasma of 20
diabetes mellitus patients (aged 27e82 years) compared to healthy
male (aged 20e27 years) non-smoking subjects: 0.47 ± 0.12 mM vs.
0.44 ± 0.11 mM in non-degassed plasma samples, and
0.34 ± 0.09 mM vs. 0.35 ± 0.08 mM in deoxygenated plasma samples
[81]. Niskanen and colleagues found in 93 T2DM patients
(1.00 ± 0.48 mM) and 22 subjects with impaired glucose tolerance
(1.04 ± 0.48 mM) higher plasma MDA concentrations compared to
96 subjects with normal glucose tolerance (0.75 ± 0.46 mM) [101].
Plasma MDA concentrations were found to be related to plasma
glucose and insuline levels. Also in contrast to other diseases, the
beneficial effects of anti-oxidant supplementation seem to be more
convincing. Among the anti-oxidants tested, vitamin E (a-tocoph-
erol) was found most effective in lowering lipid peroxidation in
diabetes rather than lowering protein and DNA oxidation [102]. Yet,
besides the clear reduction of lipid peroxidation there are no
convincing clinical effects of vitamin E supplementation in human
diabetes. It is worth mentioning that elevated oxidative stress in
human diabetes is also manifested on the basis of other lipid per-
oxidation biomarkers including the F2-isoprostanes. Thus, we
found higher excretion rates of 15(S)-8-iso-PGF2a in humans with
T2DM than in age-matched non-diabetic normolipidemic subjects
[103] (see also the review article [104]). Interestingly, atorvastatin
supplementation for 30 weeks did not reduce urinary excretion of
15(S)-8-iso-PGF2a, suggesting that atorvastatin, unlike vitamin E,
does not reduce oxidative stress despite its strong lipid-lowering
effect. In that study we did not observed significant changes in
TxA2 synthesis assessed by measuring its major urinary metabolite
2,3-dinor-TxB2 [103].
Compared to other anti-oxidants including N-acetylcysteine,
vitamin E seems to act anti-oxidatively and to reduce lipid perox-
idation on particular mechanisms. Interestingly, vitamin E supple-
mentation to T2DM patients has been reported to improve platelet
function and prostaglandin metabolism in diabetes mellitus [105].
In 29 children with T1DM, baseline plasma MDA (0.43 ± 0.02 vs
0.38 ± 0.04 mM) and TxB2 (1.50 ± 0.24 vs 0.92 ± 0.15 ng/mL) were
higher as compared to 21 non-diabetic children, with moderate
correlations between MDA (measured by HPLC) and TxB2 (deter-
mined by ELISA) [106] and no differences for vitamin E plasma
levels. Vitamin E supplementation (100 IU/day) but not placebo to
T1DM children for 3 months increased a-tocopherol plasma con-
centration, and decreased both MDA and TxB2 plasma concentra-
tion. Unfortunately, Jain et al. did not report whether or not plasma
MDA and TxB2 did correlate with each other in the non-diabetic
children and in the T1DM children after vitamin E supplementa-
tion [106]. These findings together with the well-established origin
of MDA and TxA2 determined suggest that diabetic patients have
23
hyperaggregable platelets and that vitamin E supplementation can
normalize synthesis of MDA and TxA2 in human platelets. The same
group has reported that vitamin E supplementation normalizes
MDA concentration in T1DM children [107].
In addition to vitamin E supplementation, the effects of u-3
PUFAs, notably eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA), alone or in combination with other anti-oxidants on
lipid peroxidation in humans have been often investigated in the
past and is still an area of research. On the basis of the reported data
for MDA in plasma, serum and urine, the effects of u-3 PUFAs
supplemented as fish oil, menhaden oil or as other type of sup-
plements are contradictory and no conclusion can be drawn,
whether or not u-3 PUFAs reduce lipid peroxidation, which is
widely a generally acceptable working hypothesis. In consideration
of the numerous scientific reports, here the remarkable paper by
Allard and colleagues appeared about 20 years ago [108] will be
discussed. The authors reported that „supplementing the diet with
u-3 fatty acids resulted in an increase in lipid peroxidation, as
measured by plasma MDA release and lipid peroxide products,
which was not suppressed by vitamin E supplementation.“ [108].
Daily intake of 6.26 g EPA and DHA for 6 weeks increased the
plasma MDA concentration from about 1.9 mM to 3.1 mM indepen-
dent of the vitamin E, whereas olive oil did not change the plasma
MDA concentration also independent of vitamin E [108]. These
results confirmed previous studies in humans and animals (dis-
cussed in Ref. [108]). The authors explained their results by
assuming that the fish oil used already contained EPA and DHA
peroxides which upon ingestion contributed to plasma MDA. An
alternative explanation could be that native fish oil-derived EPA
and DHA contributed to MDA by lipid peroxidation. It is worthy of
mention that fish oil but not olive oil supplentation increases the
fraction in phospholipids of EPA by about 800% and of DHA by 133%
[108]), and that EPA and DHA may contribute to MDA to a higher
extent than arachidonic acid (see next section). Previously, we
found that supplementation for 12 weeks of u-3 fatty acids in
combination with vitamins E and A, copper and selenium to
rheumatoid patients decreased the concentration of plasma 15(S)-
8-iso-PGF2a by 21%, whereas the reduction was 14% upon placebo
(soy oil) [109]. The higher decrease in the verum group may be due
to the supplemented u-3 fatty acids which may shift the F2- to F3-
isoprostane formation. However, because 15(S)-8-iso-PGF2a excre-
tion remained unchanged in both groups [109], the small decrease
seen in plasma 15(S)-8-iso-PGF2a may be due to the smaller age of
the plasma samples collected at the end of the 12-weeks lasting
study (see Refs. [33,39]). The potential effect of the plasma age on
lipid peroxidation is more convincing when the plasma MDA con-
centration is considered (Fig. 3). In the placebo and verum groups of
a previously in detail described study on rheumatoid arthritis pa-
tients [110], we found similar median plasma MDA concentrations
at the start of the study: 851 vs. 818 nM (P ¼ 0.44). The median MDA
plasma concentrations at the end of the study, i.e., after 12 weeks of
supplementation, was lower in the placebo group (i.e., 477 vs.
851 nM, P ¼ 0.02), but almost unchanged in the verum group (i.e.,
751 vs. 818 nM, P ¼ 0.59) (Fig. 3). These findings suggest that PUFAs
supplementation increased MDA formation, most likely due to the
elevation of the circulating EPA concentration in the patients sup-
plemented with u-3 fatty acids [110].
Gestational diabetes has a similar pathodology to T2DM. Arribas
and colleagues measured serum MDA concentrations (by HPLC as
TBA-MDA derivative) in 126 women with gestational diabetes [111].
The MDA concentration was found to differ between diabetic and
non-diabetic women at the 1st and the 2nd but not at the 3rd
trimester, indicating a higher lipid peroxidation at the 1st and 2nd
trimester (about 1.1 mM) and its normalization at the 3rd trimester
(about 0.8 mM) in gestational diabetes. Unlike in T1DM children
24 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
Fig. 3. Plasma concentration of MDA at baseline (0 weeks, 0w) after 12 weeks (12w)
co-supplementation of u-3 PUFAs with vitamins E and A, copper and selenium to
patients suffering from rheumatoid arthritis. Patients received other verum group
(VER) or placebo (PLA). Paired t-test within the groups and Mann-Whitney test were
used between the groups. Data are shown as mean ± standard error. Note the decadic
logarithmic scale on the y axis. MDA was determined in plasma by GC-MS/MS as
described elsewhere [33]. The plasma samples were collected in a study described in
detail previously [110].
[106], Arribas et al. found a weak correlation between serum MDA
and HbA1c when considered diabetic and non-diabetic pregnant
women together [111].
An important yet almost entirely underestimated effect with
regard to circulating biomolecules is that many of them are not
evenly distributed between plasma/serum and erythrocytes.
Ramírez-Zamora and colleagues found that MDA (measured by the
TBARS assay) is evenly distributed between serum and erythrocytes
in 90 healthy non-diabetic subjects; in 90 T2DM patients the serum
MDA concentration was comparable to that in non-diabetic sub-
jects (about 0.4 mM), but the MDA concentration in the erythrocytes
of the T2DM patients was almost twice the serum concentration
[112]. In healthy humans, we found by GC-MS/MS that hemolysis
contribute to plasma MDA [46]. Whether the higher MDA con-
centrations measured in the red blood cells of the T2DM patients,
which are more succeptible to hemolysis than of non-diabetic
subjects [112], are generated artefactually ex vivo has not been
investigated but may be relevant from a quantitative point of view
[46].
It must be pointed out that in the far majority of the articles
discussed above no description of the age of the samples at the time
point of sample analysis is provided. Given the potential effect of
long-time storage of plasma/serum samples on MDA concentration,
the biological outcome of long-term term studies may be limited
and the drawn conclusions, such as co-supplememtation of vitamin
E to fish oil to reduce PUFAs-associated lipid peroxidation, may be
questioned.
5. Summary, conclusions and outlook
PUFAs including arachidonic acid (C20:4), a-linolenic acid
(C18:3) and linoleic acid (C18:2) are thoroughly distributed in hu-
man body. These lipid substances undergo many enzymatic and
non-enzymatic reactions. The lipid peroxidation reaction is likely to
start with the chemical or enzyme-catalyzed abstraction of an
allylic H atom, that is a H atom of a methylene group positioned
between two olefinic bonds of PUFAs. In the next step one molecule
of the diradical O2 attacks the radical CH moiety to form a perox-
yradical. In the case of arachidonic acid, an additional dioxygen
molecule attacks the carbon chain to produce an endoperoxide. The
final reaction products of the PUFAs peroxidation comprise per-
oxygenated species that maintain the entire carbon chain such as
PGG2 and PGH2 from arachidonic acid, as well as small fragments
such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE)
from peroxidized arachidonic acid (Scheme 7).
In vitro investigations by Bielski and colleagues with synthetic
O2
e/HO2
(pKa, 4.7) in strongly acidic or strongly alkaline aqueous
ethanolic solutions of various fatty acids, including oleic acid,
linoleic acid, linolenic acid, arachidonic acid, 9,11- und 10,12-octa-
decadienoic acids, revealed that only linoleic acid (9,12-
octadecadienoic acid), linolenic acid (9,12,15-octadecatrienoic
acids) and arachidonic acid (5,8,11,14-eicosatetraenoic acid) reac-
ted with O2
e/HO2
[113]. Unfortunately, in that study MDA, HNE
and other possible peroxidation products had not been measured.
Nevertheless, the study by Bielski et al. suggests that MDA is likely
to be formed from the O2
e/HO2
einduced peroxidation of unsat-
urated fatty acids with double bonds being separated by a methy-
lene (CH2) group. This configuration seems to be an important
requirement for the formation of MDA from PUFAs with isolated
double bonds such as linoleic acid, linolenic acid, and arachidonic
acid by a mechanism involving the O2
e/HO2
system, 1,3-
cycloaddition of a superoxide group to the 1,4-olefinic system and
breakup of the carbon chain. Own investigations with
[5,6,8,9,11,12,14,15-2H8]arachidonic acid (i.e., all 8 deuterium atoms
are olefinic), recombinant ovine COX-2 and reduced glutathione
(GSH) revealed formation of singly labelled MDA, i.e., OHCe
CH2eC2HO and doubly labelled MDA, i.e., O2HC-CH2-C2HO, at
comparable amounts (Fig. 4A), as well as a mixture of 12-hydroxy-
heptadecatrienoic acid with 5 and 6 2H atoms [26] (Fig. 4B and C). In
addition to 12-hydroxy-heptadecatrienoic acid, we observed two
peaks eluting in front of 12-hydroxy-heptadecatrienoic acid which
are likely to be isomeric hydroxy-heptadecatrienoic acids (Fig. 4C).
These observations may suggest that even COX-dependent perox-
idation of arachidonic acid may occur via different mechanisms and
that thiols such as reduced glutathione (GSH), intracellularly the
most abundant endogenous anti-oxidant, may even facilitate the
formation of MDA and 15(S)-8-iso-PGF2a.
The number of MDA molecules that can maximally be formed
from PUFAs may correspond to the number of separated methylene
groups. Thus, the monounsaturated oleic acid and the saturated
stearic acid would not cycloperoxidize to subsequently decompose
to MDA and other species. On the other hand, linoleic, linolenic,
arachidonic, eicosapentaenoic acid and docosahexaenoic could
provide maximally 1, 2, 3, 4 and 5 MDA molecules per PUFA
molecule, respectively. Such a mechanism may explain the increase
in plasma MDA concentration upon supplementation of EPA- and
DHA-rich fish oil but not olive oil [108]. The close correlation be-
tween MDA and HNE in human plasma suggests that these species
can be formed simultaneously from a common peroxidized
arachidonic acid intermediate. MDA and TxB2, the stable metabolite
of TxA2, are also closely related. Their COX-1 catalyzed formation
can be inhibited by acetylsalicylic acid (aspirin). Human platelets
seem to be the major origin of MDA. There is also evidence that the
formation of the particular F2-isoprostane 15(S)-8-iso-PGF2a is
catalyzed, at least in part, by the COX in the human body (see
discussion in Ref. [26]). COX-catalyzed formation of the C3-species
MDA is accompanied by the formation of its complementary C-17
species 12-hydroxy-heptadecatrienoic acid (12-HHT) at almost
stoichiometric amounts (e.g., Ref. [18]). MDA, HNE and 15(S)-8-iso-
PGF2a are generally considered biomarker of lipid peroxidation.
 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30 25

Scheme 7. Requirement for the formation of malondialdehyde (MDA) from fatty acids by endoperoxidation. MDA is formed from endoperoxidation of unsaturated fatty acids of
which the double bonds are separated by a methylene (CH2) group. The number of possible MDA molecules corresponds to the number of such methylene groups. The mono-
unsaturated oleic acid and the saturated stearic acid cannot be peroxidized to form MDA. According to this mechanism, endoperoxidation of eicosapentaenoic acid (EPA) could
provide four MDA molecules produced on different position of the molecule (not shown).

However, the concentration of 12-HHT in plasma of healthy
humans is very low, e.g., 10 pg/mL corresponding to about 36 pM
[38]. This 12-HHT plasma concentration is comparable to that of
TxB2 and its major metabolites [114], i.e, but it is more than three
orders of magnitude lower than MDA plasma concentrations. These
quantitative considerations may suggest that simultaneous for-
mation of MDA and 4-hydroxy-2-nonenal (HNE) is much more
likely than the simultaneous formation of MDA and 12-HHT from
arachidonic acid.
Both free radicals and enzymes such as COX contribute to the
26 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
Fig. 4. GC-MS spectra of the deuterium-labelled reaction products (A) malondialde-
hyde and (B) 12-hydroxy-heptadecatrienoic acid (12-HHT) formed upon incubation of
10 mM [5,6,8,9,11,12,14,15-2H8]-arachidonic acid (d8-AA) with 10.7 nM recombinant
ovine COX-2 in the presence of 100 mM GSH for 30 min at 37  C. (C) The mass spectrum
shown in (B) was obtained from the GC peak eluting at 24.33 min. The two baseline-
separated GC peaks eluting in front of 12-HHT are presumably isomers of 12-HHT and
have the same m/z of 356. MDA and 12-HHT were analyzed as dipentafluorobenzyl
derivatives and pentafluorobenzyl ester trimethylsilyl ether derivatives, respectively.
This Figure was constructed with data reported in Ref. [26].
formation of MDA, HNE and 15(S)-8-iso-PGF2a. Their concentration
in biological samples such as plasma or serum and urine reveals the
contribution of chemicals and enzymes to the peroxidation at least
of arachidonic acid. As the extent of contribution of chemical and
enzymatic peroxidation is unknown, the concentration of MDA,
HNE and 15(S)-8-iso-PGF2a in biological samples is rather a mea-
sure of the total lipid peroxidation. From the quantitative point of
view, the concentration of MDA and HNE in human plasma/serum
and urine is about three orders of magnitude higher than that of
15(S)-8-iso-PGF2a. The comparably slightly higher MDA concen-
tration is likely to result from the concentration of their PUFAs
precursors and their succeptibility to lipid peroxidation. It seems
that MDA can be formed from arachidonic acid, a-linolenic acid and
linoleic acid, while HNE can be formed only from arachidonic acid.
In addition to the nitrated PUFAs, nitration of arachidonic acid has
been reported to isomerize arachidonic acid into four trans-arach-
idonic acid species which were measured in human plasma at a
total concentration of about 20 ng/mL corresponding to 66 nM
[115] (see Table 2).
The theoretically possible 64 F2-isoprostanes are divided into
four groups and are considered biomarkers of lipid peroxidation
[9,116]. At least for 15(S)-8-iso-PGF2a (iP F2a-III) has been demon-
strated that COX isoforms may contribute in vitro and in vivo to its
formation (see Ref. [26] and citations therein). On the assumption
that HHT isomers can be produced from arachidonic acid analogous
to the F2-isoprostanes, in total four HHT isomers could be formed:
12-HHT (type III), 9-HHT (type V), 8-HHT (type IV), and 5 HHT (type
VI) (see Scheme 8 and Table 2). Thus far, 12-HHT has been identified
and quantitated, whereas there is indication that the other three
HHT isomers are likely to be formed from COX. But, their identity
and quantity remain to be established.
MDA is a useful biomarker of lipid peroxidation. However, the
reliable determination of its concentration in biological samples,
notably in lipid-rich samples such as plasma or serum, is a formi-
dable challenge both from an analytical and a biological perspec-
tive. Many different analytical methods are available for the
measurement of MDA in biological samples. Without an exception,
all methods reported so far for MDA require its chemical conversion
to derivatives with improved physicochemical properties for
chromatographic separation and detection. The most famous
chemical derivatization uses thiobarbituric acid (TBA) and forms
the basis for the detection of MDA and other not yet identified TBA
reactive substances (TBARS). The selectivity of the TBARS is
improved by extracting the MDA-(TBA)2 derivative with n-butanol.
Yet, the highest gain of selectivity is reached by separating the
MDA-(TBA)2 derivative by HPLC prior to its visible absorbance or
fluorescent detection. This HPLC-TBARS assay is since several de-
cades the most widely used assay for MDA.
TBA and other derivatization reagents such as 2,3-dinitrophenyl
hydrazine (DNPH) are directed towards the aldehyde groups.
Another recently reported derivatization method utilizes the CH-
acidity of the two methylene H atoms of MDA. In aqueous
acetone, both H atoms of the methyle group are substituted by
pentafluorobenzyl (PFB) bromide (PFBeBr). The MDA-(PFB)2 de-
rivative has excellent chromatographic and mass spectrometric
properties and allows highly specific and sensitive quantitative
determination of MDA in biological samples. Free, non-covalently
bound/conjugated MDA is presumably the sole MDA species that
reacts with PFB-Br. In the case of TBA and hydrazine-groups con-
taining derivatization reagents for MDA, it is still unclear whether
only free, non-covalently bound/conjugated MDA is derivatized, or
many different covalently conjugated MDA species can also react
with the derivatization reagents and contribute to the analytical
outcome. Thus, reported MDA concentrations obtained by applying
different methods are likely to deviate, thus excluding dependable
comparison.
Yet, the greatest challenges in the analysis of MDA in plasma,
 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30 27

Table 2
Summary of identified and supposed arachidonic acid (AA) peroxidation products.

Name Formula Molecular mass Note

Malondialdehyde (MDA) CH2(CHO)2 72.1 From AA and other PUFAs

1,3-Octanedioic acid (CH)4(CH2)3COOH 139.1 Supposed
4-Oxo-2-nonenal (ONO) CH3(CH2)4C(¼O)CH]CHeCHO 154.2 From AA
4-Hydroxy-2-nonenal (HNE) CH3(CH2)4CH(OH)CH]CHeCHO 156.2 From AA
12-Hydroxy-5,8,10-heptadecatrienoic acid (12-HHT) C17H28O3 280.4 Type III
9-Hydroxy-5,7,11-heptadecatrienoic acid (9-HHT) C17H28O3 280.4 Type V, supposed
8-Hydroxy-5,9,11-heptadecatrienoic acid (8-HHT) C17H28O3 280.4 Type IV, supposed
5-Hydroxy-6,8,11-heptadecatrienoic acid (5-HHT) C17H28O3 280.4 Type VI, supposed
trans-Arachidonic acid C20H32O2 304.5 Four isomers
F2-Isoprostanes including 15(S)-8-iso-PGF2a (Type III) C20H34O5 354.5 Types III, IV, V, VI

Scheme 8. Simplified schematic proposing the formation of MDA, the four types of F2-isoprostanes, the four isomeric hydroxy-heptadecatrienoic acids, 4-hydroxy-2-nonenal (HNE)
and the corresponding aldehydes from the indicated endoperoxides of arachidonic acid. See also Table 2.

serum and tissue arise from pre-analytical issues. MDA, HNE and and storage time. A major contribution is the presence of molecular
15(S)-8-iso-PGF2a can be formed artefactually in plasma and serum oxygen. Plasma/serum deoxygenation by bubbling argon through
at concentrations far above the physiological concentrations. the samples is a highly effective procedure, considerably more
Artefactual formation of MDA depends on the storage conditions effective than the use of anti-oxidants/radical scavengers such as
28 D. Tsikas / Analytical Biochemistry 524 (2017) 13e30
butylated hydroxytoluene (BHT) or EDTA at mM-concentrations.
Sample storage is of particular importance in long-term experi-
mental and clinical studies investigating the progression of disease,
the effects of supplementation of drugs or anti-oxidants, the effects
of smog in areas of high population density and industry, nutrition,
smoking, and other forms of lifestyle including physical excerise
and sports. Blood sampling has been reported to compromise
measurement of MDA, yet the reported effects are contradictory
apparently depending on the analytical method used and the time
elapsed from blood sampling until sample work up and MDA
analysis.
Based on MDA measurement, mostly by applying the TBARS
assay to plasma or serum samples, numerous diseases have been
associated with elevated lipid peroxidation. Yet, thus far there is no
convincing evidence of the proposed associations for the majority
of the investigated diseases. The reasons include mainly the above
mentioned pre-analytical factors, as well as purely analytical fac-
tors such as lacking selectivity. Human diseases with a doubtful role
of lipid perixidation include the Alzheimer's and Parkinson dis-
eases. In contrast, there is more convincing evidence that diabetes
mellitus is associated with elevated lipid peroxidation on the basis
of higher plasma/serum MDA concentrations compared to non-
diabetic humans. A potential difficulty in investigations on a role
of lipid peroxidation in diabetes mellitus is the observation that
MDA may be evenly distributed between plasma/serum and red
blood cells in non-diabetics, but non-evenly in diabetes with MDA
accumulating in the erythrocytes. This recent observation is worth
of further investigation, also considering the fact that hemolysis
may artefactually contribute to MDA. There is increasing evidence
that in diabetes mellitus lipid peroxidation and other kinds of
oxidative stress such as nitrosative stress are not the result of dia-
betes mellitus, but rather oxidative stress is causally responsible for
diabetes due to the high succeptibility of pancreatric b-cells to
oxidative stress, which is believed to be due to a low anti-oxidant
defence potential of these cells, for instance compared to red
blood cells.
There are numerous reported intervention studies with drugs
and anti-oxidants which have generated contradictory results. The
most dependable results were obtained from the use of vitamin E,
which also substantiated the close relationship between MDA and
TxA2 from human plateletes. Aspirin inhibits concomitantly MDA
and TxA2 synthesis in human platelets and reduces platelet
aggregability. The effects of other drugs that may influence platelet
function need to be considered when drawing conclusions from
experimental and clinical studies.
An appreciable drawback in the area of oxidative stress is the
absence of suitable animal models of oxidative stress. Available and
widely used animal models are based on the use of paracetamol
(acetaminophen) or carbon tetrachloride (CCl4). Yet, these models
are too invasive and hazardous, because the oxidants must be used
at very high doses. Both chemicals may induce oxidative stress
when measured as MDA and/or 15(S)-8-iso-PGF2a, but, at the same
time, they poison the animals. Both these models lack dependable
pharmacokinetics. In humans, there is no model of oxidative stress
thus far. The utility of the recently proposed human model of
oxidative stress, which is based on a single oral ingestion of a 500-
mg paracetamol tablet by adults, remains to be demonstrated by
measuring the newly proposed biomarkers of oxidative (di-para-
cetamol) and nitrosaive stress (3-nitro-paracetamol), in addition to
established biomarkers of oxidative stress including MDA in
healthy and disease humans. A single oral dose of about 10 mg
paracetamol/kg body weight should also be suitable in chlidren.
In summary, peroxidation of free and esterified PUFAs generates
numerous reaction products. MDA and HNE are evidently products
of chemical and enzymatic lipid peroxidation of physiological
PUFAs including arachidonic acid (Table 2). From an analytical point
of view, quantitative determination of MDA in plasma, urine and
other biological samples is more easier than that of HNE because of
its hydroxy group that needs a separate derivatization step when
analyzed by GC-MS. MDA and HNE correlate closely with each
other. A major portion of biological MDA derives from arachidonic,
whereas presumably every HNE molecule stems exclusively from
arachidonic acid. Measurement of MDA alone is likely to provide
the same information about lipid peroxidation as HNE, HHT or
15(S)-8-iso-PGF2a. Yet, reliable measurement of MDA particularly in
plasma and serum is challenging for every analytical technique and
requires special precautions at the pre-analysis stage. For the sake
of comparability of results and informational value of MDA, HNE,
HHT and/or 15(S)-8-iso-PGF2a as measures of lipid peroxidation in
experimental and clinicals studies, scientific reports must include
detailed information about the used analytical and pre-analytical
conditions. Special emphasis should be given to the description of
blood coagulation/anti-coagulation, storage conditions, storage
time and age of the samples at the time point of analysis.
Acknowledgment
Prof. Andreas Hahn (Institute of Food Science and Human
Nutrition, Leibniz University of Hannover, Hannover, Germany) is
gratefully thanked for providing plasma samples from a previous
study from his group [110] for the measurement of malondialde-
hyde in the author's laboratory. The author is grateful to former
colleagues from the Institute of Clinical Pharmacology of the
Hannover Medical School for donating blood in the circadian
rhythm study.
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