J. Phycol.

48, 13621373 (2012)

2012 Phycological Society of America
DOI: 10.1111/j.1529-8817.2012.01208.x

ESTIMATION OF LIPID HYDROPEROXIDE LEVELS IN TROPICAL

MARINE MACROALGAE1
Puja Kumari, Ravindra Pal Singh, A. J. Bijo, C. R. K. Reddy,2 and Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, 364002,
Gujarat, India

index; FA, fatty acid; FAME, fatty acid methyl ester;

FOX, ferrous oxidation-xylenol orange; FOX1, ferrous oxidation-xylenol orange version 1; FOX2, ferrous oxidation-xylenol orange version 2; H2O2,
hydrogen peroxide; HPO, hydroperoxy HPLC mix;
GC, gas chromatography; GCMS, gas chromatography
mass spectrometry; LCMS, liquid chromatography
mass
spectrometry;
LHPOs,
lipid
hydroperoxides; MDA, malondialdehyde; PC1, principal
component 1; PC2, principal component 2; PCA,
principal component analysis; POD, peroxidase;
PUFA, polyunsaturated fatty acid; ROS, reactive
oxygen species; SLP, specific lipid peroxidazibility;
SOD, superoxide dismutase; TBARS, thiobarbituric
acid-reactive substances; TFA, total fatty acid; TL,
total lipid; TPP, triphenylphosphine

The incipient levels of lipid hydroperoxides

(LHPOs) were determined in selected green, brown,
and red macroalgae by the FOX assay using
hydroperoxy HPLC mix. The LHPOs contents varied
between the investigated species and showed
relatively low values in this study. Among the greens,
it varied from 12 6.2 lg
g1 (Codium sursum) to
31.5 2.8 lg
g1 (Ulva lactuca), whereas in reds,
from 5.7 1.6 lg
g1 (Gracilaria corticata) to
46.2 6 lg
g1 (Sarconema filiforme), and in browns,
from 4.6 4.4 lg
g1 (Dictyota bartayresiana) to
79 5.0 lg
g1 (Sargassum tenerrimum), on fresh
weight basis. These hydroperoxides represented a
minor fraction of total lipids and ranged from 0.04%
(S. swartzii) to 1.1% (S. tenerrimum) despite being a
rich source of highly unsaturated fatty acids. The
susceptibility of peroxidation was assessed by specific
lipid peroxidazibility (SLP) values for macroalgal
tissues. The LHPO values were found to be
independent of both the PUFAs contents and their
degree of unsaturation (DBI), as evident from the
PCA analysis. SLP values were positively correlated
with the LHPOs and negatively with DBI. The FOX
assay gave  20-fold higher values for LHPOs as
compared to the TBARS method for all the samples
investigated in this study. Furthermore, U. lactuca
cultured in artificial seawater (ASW) enriched with
nutrients (N, P, and NP) showed a sharp decline in
LHPOs contents relative to those cultured in ASW
alone P  0.05. It is inferred from this study that the
FOX assay is an efficient, rapid, sensitive, and
inexpensive technique for detecting the incipient
lipid peroxidation in macroalgal tissues.

Lipid peroxidation is a well recognized indicator

of oxidative stress in all types of organisms, including plants, bacteria, fungi, algae, and mammalian
systems. In addition, their products exhibit a wide
variety of biological and cell signaling functions
(Spickett et al. 2010). Lipid peroxidation can be
either enzymatic mediated by lipoxygenases and
cyclooxygenases, or nonenzymatic free radical
mediated, where several chain reactions are mediated by reactive oxygen species (ROS), which
themselves are unavoidable consequences of aerobic
metabolism.
Although lipid peroxidation is a well-investigated
phenomenon, lipid hydroperoxides (LHPOs) have
received comparatively less attention than the other
lipid oxidized products because of their either instability or lengthy procedures involving of expensive
and sophisticated equipments for detection. The
LHPOs have been estimated by several analytical
approaches, such as high-performance liquid
chromatography (HPLC; Nakamura and Maeda
1991, Hui et al. 2005), gas chromatography (GC; Turnipseed et al. 1993), electrospray mass spectrometry
(Spickett et al. 1998), iodine oxidation (Jessup et al.
1994), heme degradation of peroxides (Frei et al.
1998), cyclooxygenase activation (Pendleton and
Lands 1987, Calzada et al. 1997), chemiluminescence
detection (Yamamoto 1994), immunological assays
(Paradis et al. 1997), conjugated diene measurement
(Moore and Roberts 1998), and thiobarbituric

Key index words: fatty acid; FOX; lipid hydroperoxides;

macroalgae; TBARS
Abbreviations: 12(S)-HpETE, 12(S)-Hydroperoxyeicosatetraenoic acid; 15(S)-HpETE, 15(S)-Hydroperoxyeicosatetraenoic acid; 5(S)-HpETE, 5(S)-Hydroperox
yeicosatetraenoic acid; 13(S)-HpODE, 13(S)-Hydroperoxyoctadecadienoic acid; 9(S)-HpODE, 9(S)-Hydroperoxyoctadecadienoic acid; 13-HpOTE, 13-Hydr
operoxyoctadecatrienoic acid; ANOVA, analysis of
variance; ASW, artificial seawater; BHT, butylated
hydroxytoluene; CAT, catalase; DBI, double bond
1
2

Received 29 February 2012. Accepted 5 June 2012.

Author for correspondence: e-mail: crk@csmcri.org.

1362

LIPID HYDROPEROXIDES IN MACROALGAE

acid-reactive substances (TBARS) assay (Hodges et al.

1999). Of these methods, the determination of
conjugated dienes and TBARS (malondialdehyde
estimation) methods have been explicitly used for
LHPOs estimation in various biochemical studies
(Kumar et al. 2010a,b, Kapich et al. 2011). However,
both of these methods have been debated for their
lack of specificity and accuracy because they do not
measure lipid peroxidation products exclusively, and
moreover substantial amounts of diene-conjugated
materials in tissues do not contain the hydroperoxide
functional group (Smith and Anderson 1987,
Bonnes-Tourel et al. 1992). In addition, the inappropriate modification in the assay further worsens the
reliability of the method (Lapanna and Cuccurullo
1993).
In view of this, an alternative simple and rapid
method for LHPO estimation using ferrous oxidation-xylenol orange (FOX) assay (versions 1 and 2)
has been developed (Jiang et al. 1991, Wolff 1994,
Nourooz-Zadeh et al. 1995). This technique is primarily based on the hydroperoxide-mediated oxidation of ferrous to ferric ions under acidic conditions
with subsequent binding of ferric ions to the ferricsensitive dye xylenol orange [o-cresolsulphonphthalein-3,3-bis(methyliminodiacetic acid) sodium salt]
to give a colored complex with an absorbance maximum at 560 nm. In the FOX1 assay, the reagent is
prepared in aqueous medium with sorbitol, whereas
in FOX2, the reagent contains HPLC grade methanol and butylated hydroxytoluene (BHT). Fe+2 and
H2SO4 are present in both the versions (Nouro
oz-Zadeh 1999, Banerjee et al. 2003). Furthermore,
the FOX2 method was developed based on the measurement of absorbance of the sample mixture
before and after triphenylphosphine (TPP) addition
that selectively reduces the hydroperoxides into
their corresponding alcohols. In this way, the FOX2
method is essentially made specific for LHPOs
estimation by TPP addition, hence, nullifying the
signals from interfering compounds such as phenols
(Nourooz-Zadeh 1999, Griffiths et al. 2000, Yin and
Porter 2003). Both versions of the FOX method
have been successfully employed to measure LHPOs
in various biological samples including mammalian
tissues (Firth et al. 2007), reconstructed membrane
systems (Deiana et al. 1999, Yin and Porter 2003),
LDL lipoproteins (Jiang et al. 1992), plasma (Banerjee et al. 2003, Gay and Gebicki 2003), plant tissues
(Hermes-Lima et al. 1995, Griffiths et al. 2000,
DeLong et al. 2002), edible oils (Nourooz-Zadeh
et al. 1995, Yildiz et al. 2003), food products, such
as chicken, fish, fried snacks (Grau et al. 2000,
Eymard and Genot 2003, Navas et al. 2004), mycelia
lipids (Fakas et al. 2008), and for the estimation of
lipoxygenase activity (Pinto et al. 2007, Fukuzawa
et al. 2009).
As far as macroalgae are concerned, the TBARS
method has been the choice of researchers for the
determination of lipid peroxidation in various

1363

biochemical and physiological studies (Li et al.

2010, Kumar et al. 2010a, 2011). As the TBARS
method gives an estimate of aldehyde products that
are secondary peroxidation products formed from
LHPO degradation (Spickett et al. 2010), the actual
level of LHPOs in macroalgae is largely unknown
except for the studies of Bouarab et al. (2004) and
Gaquerel et al. (2007). These authors estimated the
levels of linoleate, linolenate, and arachidonate hydroperoxides, such as 13-hydroperoxyoctdecadienoic
acid (13-HpODE), 13-hydroperoxyoctdecatrienoic
acid (13-HpOTE), and 12 (S)-hydroperoxyeicosatetraenoic acid (12 (S)-HpETE), by GCMS and LC
MS in Chondrus crispus subjected to biotic and
methyl jasmonate stress, respectively. The application of the FOX assay has been limited to the assessment of antioxidant potential of a mycosporine-like
amino acid, porphyra-334 isolated from Pyropia yezoensis (Tao et al. 2008). However, to the best of our
knowledge, there is only a single report of LHPOs
determination in the unicellular alga Chlamydomonas
reinhardtii using the FOX2 assay by Griffiths et al.
(2000).
Tropical macroalgae are exposed to high extremes
of abiotic factors, such as temperature, UVR, salinity,
desiccation, and nutrients (carbon, nitrogen, and
phosphorus) limitation. These extreme conditions
lead to ROS formation that may have deleterious
effects on cell membranes causing lipid peroxidation
(Collen and Davison 2001, Kumar et al. 2010b,
Lesser 2012). However, despite being continuously
exposed to different biotic and abiotic stresses in
their natural habitat, macroalgae maintain an intricate redox balance between lipid peroxidation and
antioxidant defense system by accumulating antioxidants and up-regulation of antioxidant enzymes
(Matsukawa et al. 1997). Moreover, macroalgae are
rich sources of polyunsaturated fatty acids (PUFAs)
that are susceptible to oxidation by ROS, generating
lipid peroxides by a chain-reaction mechanism. It is
generally believed that lipid peroxidation increases
with the PUFA content and the degree of fatty acyl
unsaturation (Girotti 1998). Thus, it is quite interesting and informative to study the incipient LHPOs
levels in such a complex heterogeneous systems as
macroalgae which are natural sources of numerous
biologically active antioxidants and secondary
metabolites.
Ulva sp. are commercially important green macroalgae and are emerging as one of the model organisms for investigating complex metabolic networks
(Gonzalez et al. 2010, Dong et al. 2012, Hsu and
Lee 2012, Kerrison et al. 2012). Ulva lactuca inhabits
the intertidal region where the availability of
nitrogen (N) and phosphorus (P) in ocean varies
dramatically on spatial and temporal scales, limiting
the primary productivity over large areas. The rapid
growth of this alga has been attributed to its
high photosynthetic rates and high nutrient
uptake capacity (Naldi and Wheeler 2002). Several

1364

PU J A K U M A R I E T A L .

experiments have been undertaken to study the

uptake and assimilation of N and P comprehend
with growth response, pigment analysis, mineral
composition, and respective key enzymes (nitrate
reductase and acid/alkaline phosphatases) in different species of Ulva (Lee et al. 2005, Taylor et al.
2006, Robertson-Andersson et al. 2009, Ale et al.
2010, Naixing et al. 2012). But the level of LHPOs
caused by nutrient stress is not yet known.
In this work, we systematically studied the incipient levels of LHPOs in different green, brown, and
red macroalgal samples collected along the Gujarat
coast, India, using the FOX2 assay to correlate the
sensitivity of PUFAs to peroxidation and LHPO levels in macroalgal tissues. In these analyses, a commercial hydroperoxy HPLC mix (HPO mix) was
used for calibration and calculation of LHPO levels
and compared with the reference standard H2O2
for validation of our approach. A comparative study
of TBARS and FOX2 assay was also accomplished to
assess the suitability of the method. Furthermore,
LHPO levels were also determined in a selected common green macroalgae of Indian waters, U. lactuca,
cultured with different nutrients in the laboratory to
gain an insight into LHPO levels and their
implications in correlations with nutrient stress.
MATERIALS AND METHODS

Chemicals. Ammonium ferrous sulfate, butylated hydroxytoluene (BHT), xylenol orange [o-cresol-sulphonphthalein-3,
3-bis (methyliminodiacetic acid sodium salt)], triphenylphosphine (TPP) were purchased from Fisher Scientific,
India. Hydroperoxy HPLC Mix that contained 12(S)-HpETE,
15(S)-HpETE,
5(S)-HpETE,
and
9(S)-HpODE
and
13(S)-HpODE (5 lg each in 250 lL of final volume) was purchased from Cayman chemicals (USA). All reagents were of
the highest purity available and the solvents were of HPLC
grade.
Preparation of FOX2 reagent. FOX2 reagent was prepared
according to Griffiths et al. (2000) by dissolving xylenol orange
and ammonium ferrous sulfate in 250 mM H2SO4 to final concentrations of 1 mM and 2.5 mM, respectively. One volume of
this concentrated reagent was added to 9 volumes of methanol
containing 4.4 mM BHT to make the working FOX2 reagent
comprising of 250 lM ammonium ferrous sulfate, 100 lM xylenol orange, 25 mM H2SO4, and 4 mM BHT in 90% (v/v)
methanol.
Algal samples. All the algal species were collected in triplicates from the Saurashtra coast of Gujarat, India during
MarchApril 2010. Ulva lactuca Linnaeus, U. fasciata Delile,
U. taeniata (Setchell) Setchell & N. L. Gardner, U. flexuosa
Wulfen, Chamaedoris
auriculata Brgesen, Gracilaria corticata (J. Agardh)
J. Agardh, G. dura (C. Agardh) J. Agardh, G. salicornia (C.
Agardh) E. Y. Dawson, Sarconema filiforme (Sonder) Kylin,
Sargassum tenerrrimum J. Agardh, S. cinereum J. Agardh, Padina
tetrastomatica Hauck, and Spatoglossum asperum J. Agardh were
collected from Veraval coast (N 20 54.87, E 70 20.83), Ulva
reticulata Forsskai, U. beytensis Thivy & Sharma, Codium dwarkense Brgesen, C. sursum Kraft & A. J. K. Millar, Dictyota
hauckiana Nizamuddin, D. ciliolata Sonder ex Kutzing, D. bartayresiana J. V. Lamouroux, Sargassum carpophyllum J. Agardh,
and Sargassum sp. were collected from Kalubhar island (N

22 26.21, E 69 35.12), S. johnstonii Setchell & N. L. Gardner from Okha (N 22 93 28.42, E 69 03.58), S. plagiophyllum C. Agardh from Shivrajpur (N 22 19.87, E 94
68 56.95), and S. swartzii C. Agardh from Porbandar (N
21 38.24, E 69 35.81). The samples were immediately
transported to the laboratory in wet towels in an ice box,
then cleaned thoroughly to remove the epiphytes and other
undesired foreign matter from the fronds, frozen in liquid
nitrogen, and stored at 40C until the analysis.
For nutrient stress experiments, U. lactuca thalli were
acclimatized in artificial seawater (ASW) at 25 1C temperature under daylight white fluorescent lamps at
15 lmol photon
m2
s1 irradiance with a 12:12 h light:
dark photoperiod for 10 d. Thereafter, algal thalli were cultured with different nutrient sources, N (10 mg
L1
NaNO3), P (20 mg
L1 Na2HPO4.H2O), NP both, and
ASW alone for 7 d, as optimized for maximum growth in
this Ulva sp. in the laboratory (details provided in Supporting Information). We aimed to study the effect of different
nutrient sources on growth rate, pigments, minerals, lipid composition, and other biochemical parameters (unpublished
data). The media was changed every other day to replenish
nutrient loss. Thereafter, the tissues were frozen in liquid nitrogen and stored at 40C until the analysis.
Lipid extraction and FOX assay. Total lipids were rapidly
extracted from algal tissues by a modified Bligh and Dyer
method according to Griffiths et al. (1997). All procedures
were performed in dim light at 4C using chilled solvents
containing BHT (0.01% w/v). Fifty mg fresh weight macroalgal tissues were ground in liquid nitrogen, and lipids were
immediately extracted with chloroform/methanol (1:2, v/v,
750 lL) and 0.15 M acetic acid (100 lL) by vortexing
(2 min) and centrifuging at 2057 g for 15 min (9 2) in 2-mL
eppendorf tubes (Eppendorf, USA). The extracts were combined, and phase separation was done by adding distilled water
(1 mL) and facilitated by centrifugation at 2057 g for 5 min.
The lower chloroform phase containing lipids was collected
and evaporated under N2. The samples were resuspended in
methanol either in 100 lL for samples without TPP or in a mixture of 90 lL methanol and 10 lL TPP (25 mM in methanol)
for the FOX assay. The samples were incubated at room temperature for 30 min in the dark followed by 1 h incubation after the
addition of working FOX2 reagent. The absorbances were
determined spectrophotometrically at 560 nm, and the concentration of LHPOs was determined from the calibration
curve of hydroperoxide HPLC mix standard.
Calibration of FOX2 reagent. The calibration curves were
obtained with both the HPO mix and reference standard H2O2
for the FOX2 method for comparison and validation. TPP (in
methanol, final concentration 2.5 mM) was added to one series of samples to reduce the LHPOs to their corresponding
nonreactive hydroxide derivatives in order to authenticate the
signals generated in the samples minus TPP following the addition of FOX2 reagent. Methanol plus TPP had negligible absorbances for the two standards (LHPO mix and H2O2) and also
for both the green and red macroalgae. However, the brown
algae showed minor absorbances for methanol plus TPP
controls owing to their high polyphenolic contents that also
exhibit their absorbances in the same range 500600 nm. Furthermore, three calibration curves were generated from lipid
extracts of selected macroalgal species, namely U. lactuca,
G. corticata, and S. tenerrimum, representing each of green, red,
and brown algae, in the concentration range 10100 lL, The
linearity of each calibration curve was checked statistically with
a curve-fitting model using OriginPro8.5.1 (described in Tables
S1 and S2, see Supporting Information).
Lipid (fatty acid) quantification. Lipids were quantified as
their fatty acid methyl ester derivatives by transmethylation

LIPID HYDROPEROXIDES IN MACROALGAE

with 1 mL of 1% NaOH in methanol, followed by heating

for 15 min at 55C, adding 2 mL of 5% methanolic HCl,
and again heated for 15 min at 55C, then adding 1 mL
milli-Q water (Carreau and Dubacq 1978). Nonadecanoic
acid was used as an internal standard. FAMEs were extracted
in hexane and separated on RTX-5 fused silica capillary column, 30 m 9 0.25 mm 9 0.25 lm (Rastek) by GCMS with
helium (99.9% purity) as the carrier gas. The GCMS conditions were the same as in our earlier study (Kumari et al.
2011).
TBARS method. The level of lipid peroxidation was determined as described by Heath and Packer (1968). Algal tissue
(0.2 g) was extracted with 2 mL of 0.5% thiobarbituric acid
(TBA) prepared in 20% trichloroacetic acid (TCA), heated at
95C for 30 min, cooled in ice and centrifuged at 10,000 g for
10 min. The absorbance of the supernatant was measured at
532 nm and corrected for nonspecific absorbance by subtracting the value recorded for 600 nm. The level of lipid peroxidation was expressed as nmols of malondialdehyde (MDA)
formed using the extinction coefficient of 155 mM
cm1.
Specific lipid peroxidazibility (SLP). Specific lipid peroxidazibility was calculated as the ratio of LHPOs and double bond
index (DBI) of polyunsaturated fatty acids [linoleic (C18:2
n6), c-linolenic (C18:3 n6), a-linolenic (C18:3 n3), stearidonic (C18:4 n3), dihomogamma-linolenic (C20:3 n6),
arachidonic (C20:4 n6), eicosapentaenoic (C20:5 n3), and
docosahexaenoic acids (C22:6 n3)] per mg of lipid (Fakas
et al. 2008) to evaluate the peroxidazibility of lipids. Double
bond index (DBI) was calculated using the formula:

DBI 2  %C18 : 2 3  %C18 : 3

3  %C20 : 3 4  %C20 : 4
5  %C20 : 5 6  %C22 : 6=100
Statistical analysis. All the analyses were performed in triplicates, and mean values were reported. The LHPO values
and fatty acid contents obtained for different seaweeds were
compared by analysis of variance (ANOVA), with values significant at P  0.05. Principal component analysis (PCA) was
accomplished using Unscrambler X (Camo, USA) to study
the distribution of LHPOs among green, red, and brown
macroalgae and to decipher a relation between the susceptibility of PUFAs to peroxidation, LHPOs, and double bond
index (DBI).
RESULTS

LHPO levels in macroalgae. LHPO contents in

macroalgal tissues varied among different species and
ranged from 12 6.2 lg
g1 (Codium sursum) to
31.5 2.8 lg
g1 (U. lactuca) for green algae,
5.7 1.6 lg
g1 (Gracilaria corticata) to 46.2 6 lg

g1 (Sarconema filiforme) for red algae, and from
4.6 4.4 lg
g1 (Dictyota bartayresiana) to 79
5.0 lg
g1 (Sargassum tenerrimum) for brown algae
(P  0.01) on fresh weight basis as calculated from
the calibration curve of HPO mix (Table 1). Interestingly, the LHPO contents expressed in terms of H2O2
equivalents using the extinction coefficient of H2O2
was found to be 1.4 times higher than those values
calculated from the calibration curve of LHPO mix
(Table 1). Moreover, the LHPOs determined by the
TBARS method gave  20-fold lower values for the
investigated samples except G. corticata and D. bart-

1365

ayresiana, where this difference was relatively less, 8fold and 11-fold lower, respectively (Table 1).
However, these hydroperoxides accounted to minor
fractions of total lipids and accounted for a marginal
increase from 0.04% (S. swartzii) to 1.1% (S. tenerrimum) and 1% and 2% of TFAs contents in all the species except Sarconema filiforme, Spatoglossum asperum,
and a few Sargassum sp. (Table 1).
PUFA composition and specific lipid peroxidazibility.
Linoleic, c-linolenic, a-linolenic, stearidonic, dihomogamma-linolenic, arachidonic, eicosapentaenoic,
and docosahexaenoic acids were the major PUFAs
detected in different macroalgae. The green algae
showed higher contents of C18 PUFAs, red algae
showed higher contents of C20 PUFAs, and brown
algae contained appreciable amounts of both C18
and C20 PUFAs (Table 2). The specific lipid peroxidazibility varied with the species, their LHPO
contents and double bond indices with Sargassum
swartzii accounting for the highest SLP value
(116.1 17 lg
1; Table 2). PCA analysis performed on the data matrix, including LHPOs,
LHPO/TL, LHPO/TFA, SLP, DBI, and PUFAs,
satisfactorily explained 79% of the variations inherent in the data set; PC1 accounted for 50% and
PC2 for 29% of the variability. A strong correlation
was found between LHPOs, LHPO/TL LHPO/TFA
and SLP, whereas SLP was negatively correlated
with DBI, revealing an inverse relation as apparent
from their positions on PCA bi-plot (Fig. 2). DBI
and PUFA also showed a strong correlation indicating that DBI increases with the increase in PUFAs
contents. Both the DBI and PUFAs were independent of LHPOs, LHPO/TL, and LHPO/TFA as
they were explained by different principal components and thus were orthogonal in nature. However, statistically no significant relation was evident
for the distribution of LHPOs at the phyla level
among green, red, and brown macroalgae from the
PCA bi-plot. Instead, the y-axis separated all of the
Sargassum spp. into one group (encircled) due to
their higher loadings for LHPO, LHPO/TL,
LHPO/TFA, and SLP and Gracilaria spp., Ulva spp.
(except U. lactuca), Dictyota spp. (except D. hauckiana) to another group due to their lower loadings
for these variables (Fig. 2), indicating that LHPO
contents are species specific.
LHPO contents in U. lactuca cultured under different
nutrients. The LHPO contents in U. lactuca thalli
cultured under different nutrient sources varied significantly from the thalli cultured with artificial seawater (ASW) alone considered as control. The
control thalli showed the maximum content of
LHPOs (44 10 lg
g1 f.w.) revealing its nutrient
depleted condition. However, there was a sharp
decrease in LHPO contents in thalli supplemented
with nutrients. The thalli cultured with ASW + NP
revealed a maximum of 1.7-fold decrease in
LHPO levels followed by nitrogen supplemented
(ASW + N) thalli that reported a 1.6-fold decrease

am

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

S. no.

31.5
19.2
18.8
18.2
19.3
21.5
17.7
12
13.7
5.7
9.9
8.6
46.2
48
54.3
59
26.7
26.3
79
48.3
41.7
8
4.6
8.5
33.5

2.8efg
2.7hij
2.2hijk
4.1hijkl
6.4hij
3.3ghi
6.5hijkl
6.2ijklm
5.1ijklm
1.6m
0.8jklm
1.1klm
6cd
2.0cd
2.1bc
1.0b
4fgh
2.5fgh
5.0 a
4.2cd
1.2de
1.0lm
4.4m
0.5klm
0.85ef

0.0005de
0.0002gh
0.001gh
0.0005
0.0007hi
0.0002hi
0.0002hi
0.0005hi
0.0005hi
0.0004hi
0.0002gh
0.0002gh
0.0001c
0.0002c
0.0001fg
0.0002 b
0.0001i
0.0004de
0.0007a
0.0005cd
0.0001ef
0.0001hi
0.0005hi
0.0001hi
0.0001gh

LHPO/TL

0.005
0.002
0.002
0.002
0.001
0.001
0.001
0.001
0.001
0.001
0.002
0.002
0.007
0.007
0.003
0.009
0.0004
0.005
0.011
0.006
0.004
0.001
0.001
0.001
0.002

Values in a column are significantly different at P  0.05.

Ulva lactuca
Ulva fasciata
Ulva taeniata
Ulva flexuosa
Ulva reticulata
Ulva beytensis
Codium dwarkense
Codium sursum
Chamaedoris auriculata
Gracilaria corticata
Gracilaria dura
Gracilaria salicornia
Sarconema filiforme
Sargassum johnstonii
Sargassum cinereum
Sargassum carpophyllum
Sargassum swartzii
Sargassum sp.
Sargassum tenerrimum
Sargassum plagiophyllum
Dictyota hauckiana
Dictyota ciliolata
Dictyota bartayresiana
Padina tetrastomatica
Spatoglossum asperum

Macroalgal samples

LHPOs (lg
g1 f.w.)

calculated
from calibration
curve of hydroperoxide mix

0.03
0.02
0.02
0.02
0.02
0.02
0.02
0.01
0.01
0.01
0.02
0.02
0.12
0.24
0.16
0.1
0.01
0.07
0.2
0.24
0.08
0.01
0.01
0.02
0.07

0.003f
0.003fg
0.012fg
0.004fg
0.013fg
0.004fg
0.006fg
0.006 g
0.005g
0.004g
0.002fg
0.003fg
0.001d
0.008a
0.006c
0.002gh
0.001g
0.006e
0.013b
0.02a
0.002 e
0.002g
0.007 g
0.001fg
0.002e

LHPO/TFA

TABLE 1. Lipid hydroperoxide levels in macroalgal samples expressed as mean SD (n = 3).

1.3
0.8
0.8
0.8
0.8
0.9
0.7
0.5
0.6
0.2
0.4
0.4
1.9
2
2.3
2.45
1.1
1.1
3.3
2
1.7
0.33
0.2
0.4
1.4

f.w.)

0.11efg
0.11hij
0.51hij
0.2 hij
0.53hij
0.15ghi
0.27
0.26ijkl
0.21ijkl
0.07l
0.03jkl
0.05jkl
0.02cd
0.07 bcd
0.08bc
0.04 bcd
0.16fgh
0.11fgh
0.21a
0.17bcd
0.04de
0.04kl
0.18l
0.01jkl
0.03ef

(lmol
g

1

[44.7
[27.2
[26.7
[25.8
[27.4
[30.1
[24.6
[16.9
[19.1
[8
[14
[12.2
[65.3
[67.3
[77.1
[83.3
[37.7
[37.8
[112
[68.7
[58.9
[11.2
[6.5
[12
[47.4

[lg
g
f.w.]

3.9 efg]
3.8hij]
1.73hijk]
5.7hijkl]
2hij]
5ghi]
5hijjklm]
8 ijklm]
7.2ijklm]
2.3m]
1.1jklm]
1.6klm]
8cd]
2.3cd]
2.9bc]
1.4b]
5.4fgh]
3.6fgh]
7.2a]
5.9bcd]
1.4 de]
1.2 lm]
6.2m]
0.7klm]
1.1ef]

1

LHPOs expressed as H2O2 equivalents

(eH2O2 54660 M1
cm1)

0.026
0.025
0.02
0.018
0.026
0.027
0.019
0.016
0.018
0.02
0.017
0.017
0.03
0.024
0.022
0.029
0.019
0.02
0.038
0.03
0.035
0.012
0.012
0.011
0.023

f.w.)

TBARS

0.001bcde
0.001cde
0.002
0.001ghi
0.001bcde
0.001bcd
0.001ghi
0.001ij
0.001hi
0.00fghi
0.001i
0.001i
0.004b
0.001def
0.004efgh
0.001bc
0.001ghi
0.001fghi
0.002a
0.001b
0.002a
0.002jk
0.002jk
0.0003k
0.0001defg

(lmol
g

1

0.87
0.84
0.67
0.61
0.88
0.93
0.65
0.54
0.61
0.68
0.58
0.58
1.04
0.83
0.75
0.99
0.65
0.69
1.3
1.0
1.2
0.4
0.4
0.4
0.78

0.05cde
0.05de
0.06ghi
0.01hi
0.13cde
0.06bcd
0.04ghi
0.02ij
0.01hi
0.06ghi
0.02hi
0.01hi
0.15b
0.01def
0.13efg
0.02bc
0.02ghi
0.01fgh
0.07a
0.04bc
0.06a
0.07j
0.07j
0.01j
0.01efg

[lg
g1 f.w.]

1366
PU J A K U M A R I E T A L .

Ulva lactuca
2.4 0.2efgh
Ulva fasciata
10.6 6.2a
Ulva taeniata
1.2 0.6a
Ulva flexuosa
2.4 0.3efgh
Ulva reticulata 0.6 0.2gh
Ulva beytensis
0.2 0.2h
Codium
0.8 0.2gh
dwarkense
Codium
2.1 0.3fgh
sursum
Chamaedoris
5.4 0.4bcd
auriculata
Gracilaria
1.1 0.2h
corticata
Gracilaria
5.6 0.8bc
dura
Gracilaria
0.6 0.3gh
salicornia
Sarconema
7.8 3.8b
filiforme
Sargassum
1.0 0.4fgh
johnstonii
Sargassum
n.d.
cinereum
Sargassum
5.0 3.5cde
carpophyllum
Sargassum
3.0 0.1cdefg
swartzii
Sargassum sp.
2.3 0.5fgh
Sargassum
2.8 0.6defgh
tenerrimum
Sargassum
1.2 0.2fgh
plagiophyllum
Dictyota
0.7 0.1gh
hauckiana
Dictyota
3.6 0.1cdef
ciliolata
Dictyota
1.2 0.2fgh
bartayresiana
Padina
2.5 0.7efgh
tetrastomatica
Spatoglossum
0.1 0.02h
asperum

C18:2(n 6)

n.d.
n.d.
n.d.

0.6 0.2bc
0.1 0.03d

n.d.

0.2 0.01d

0.9 0.1

n.d.

2.0 0.5a

n.d.

0.2 0.02d

n.d.

n.d.

0.8 0.5b

0.2 0.01d

n.d.

0.1 0.01d

n.d.
n.d.

n.d.

0.1 0.01d

n.d.
n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.0 0.7bc

0.7 0.2cd

0.4 0.1efg

0.8 0.16e

0.2 0.1fg

4.5 0.15cd 0.4 0.1efg

1.8 0.4cde

1.9 0.1

11.7 0.95
b

0.3 0.1efg

0.2 0.02fg

23.0 6.0a

3.0 0.3cde

2.4 0.6cde 0.4 0.02efg

3.1 0.98cde 0.4 0.03efg

n.d.

11.6 8.1b

0.1 0.12f

0.2 0.1fg

0.1 0.01g

n.d.

1.3 0.1b

3.7 0.7cde

n.d.

0.6 0.1de

0.1g
0.5g
0.1g
0.03g
0.01g
0.01g
0.1g

0.4 0.2d

0.4 0.07d

0.9 0.3c

5.4 0.5

1.6 1.2b

0.2 0.03d

0.1 0.01g

0.3g
0.3ef
0.1g
0.01g
0.02g
n.d.
0.1 0.01g
0.4
0.5
0.2
0.1
0.1

C20:5(n 3)

3.1 2.2b

2.7 0.9bc

0.5 0.1ef

1.9 0.3

bcd

7.5 2.2a

2.0 0.8

0.5 0.2ef

2.8 0.5efg 0.3 0.04f

1.5 0.3fg

6.6 0.4

cde

9.3 2.8bc

4.2 0.3defg 1.5 0.3cdef

1.7 0.2cde
2.4 0.6bc

5.6 1.2cdef 2.4 0.7bc

14.7 10a

0.4 0.1g

5.3 2.2cdef 1.8 1.0cde

n.d.

n.d.

2.3 0.1fg
1.6 0.3fg

n.d.

6.7 1.1cde

3.1 0.3defg 0.7 0.1def

3.6 0.5defg 2.5 0.5bc

0.8 0.1g

0.4
0.9
0.2
0.3
0.1
0.1
0.3

C20:4(n 6)

0.3 0.02d 7.0 1.3cd

0.7 0.15cd 12.4 2.7ab

0.2 0.01d

0.7 0.3cd

0.1 0.05e

0.3 0.03d

n.d.

n.d.

0.2 0.05d

n.d.

0.2 0.01fg

0.1 0.01g

n.d.

2.5 1.4cde

C20:3(n 3)

0.01fg 0.3 0.03d

0.1efg
0.5 0.2cd
0.01g
0.1 0.02e
0.01g
n.d.
n.d.
n.d.
n.d.
0.02 0.01f
g
0.1 0.01
n.d.
0.2
0.3
0.1
0.1

C20:3(n 6)

n.d.

n.d.

n.d.

1.7 0.7cde

1.0 0.4b

5.8 0.6bc

0.1 0.05f

0.15de
3.1c
0.35e
0.11e
1.1de
0.2e
0.03f

0.3 0.02c 10.9 0.8a

C18:4(n 3)

1.3
5.1
0.6
0.8
0.9
0.8
0.1

C18:3(n 3)

0.1d
6.7 0.3bc
0.4b 35.4 21.5a
0.5bc 3.0 1.2c
0.01d 8.3 0.8bc
0.01d 1.4 0.03c
n.d.
2.4 0.3c
0.1 0.01d 1.0 0.3d
0.2
0.7
0.6
0.2
0.1

C18:3(n 6)

PUFAs
(lg
mg1 lipid)

A Double bond index = [2 9 (%C18:2) + 3 9 (C18:3) + 3 9 (C20:3) + 4 9 (C20:4) + 5 9 (C20:5) + 6 9 (C22:6)]/100.

ak
Values in a column are significantly different at P  0.05.

25

24

23

22

21

20

18
19

17

16

15

14

13

12

11

10

1
2
3
4
5
6
7

S. no.

Macroalgal
samples

0.2b
1.3a
0.1c
0.01c
0.02c
0.05c
0.02c
0.9
1.9
1.5
1.6
1.4
1.9
0.8

0.03ghi
0.06ab
0.02abcdefg
0.02abcde
0.4bcdeg
0.05ab
0.08hi

DBIA
()

n.d.

n.d.

n.d.

0.9 0.06ghi

1.3 0.06cdef

1.0 0.03efgh

abcd

1.8 0.06abc

1.1 1efgh

1.2 0.01defgh
1.2 0.06defgh

0.2 0.2j

1.4 0.35bcdef

1.0 0.06efgh

1.5 0.2abcdefg

1.2 0.04defgh

0.3 0.01j

1.6 0.04abcdef

0.4 0.04i

0.4 0.08 1.7 0.03

n.d.

n.d.

n.d.
n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.1 0.04c 1.6 0.06abcdef

0.1 0.04c 1.5 0.13abcdefg

1.2
2.2
0.3
0.1
0.2
0.2
0.1

C22:6(n 3)

TABLE 2. Polyunsaturated fatty acid composition and specific lipid peroxidazibility of macroalgal samples, data expressed as mean SD (n = 3).

3.1efg
1.4jk
8.1jk
2.5jk
8.6ij
1.8jk
7.7hi

36.4 0.8def

6.3 0.4jk

4.7 4.6jk

4.6 0.6k

23.8 0.7

42.1 3.6de

22.5 2.2h
65.7 4.2b

116.1 17a

43.5 0.7d

52.4 2c

32.1 1.4fg

40.0 0.5def

26.1 3.5gh

6.1 0.5jk

12.6 3.6jk

8.6 3.2jk

7.8 4.1jk

34.3
9.9
12.5
11.2
13.4
11.3
21

SLP
(lg
1)

LIPID HYDROPEROXIDES IN MACROALGAE

1367

1368

PU J A K U M A R I E T A L .

and phosphorus supplemented (ASW + P) thalli

that showed a 1.4-fold decrease (P  0.05;
Table 3). The same trend was also supported by
their SLP values that decreased with the nutrient
supplementation, with ASW + NP thalli reporting
the lowest value (2.6-fold decrease; Table 4). The
LHPO/TL ratio was also highest for the thalli cultured with ASW followed by those supplemented
with N, then NP and P, due to the higher accumulation of total lipids in ASW + P medium as compared to the ASW + N or ASW + NP treated algal
samples. More interestingly, these values as calculated from the calibration curve of LHPO mix were
again 1.4 times lower than the LHPO contents
expressed in terms of H2O2 equivalents (Table 3),
thus confirming the above mentioned trends
observed for different macroalgal samples. Similarly,
the levels of LHPOs determined by the TBARS assay
were also  20-fold lower as compared to the FOX2
method (P  0.05; Table 3).

antioxidant enzymes, and thus FOX method became

popular for determination of incipient lipid peroxidation (Griffiths et al. 2000, Banerjee et al. 2003,
Fukuzawa et al. 2006, Fakas et al. 2008). Conversely,
many researchers have also pointed toward the nonstoichiometric relationship between the amount of
hydroperoxides and Fe3+ produced as the major
drawback of the FOX methods (versions 1 and 2)
because it has been observed that linoleic acid
hydroperoxides produce twice the amount of ferric
ions per molecule as compared to the linolenic or
arachidonic acid hydroperoxides amounts (Gay and
Gebicki 2002, Vega et al. 2005, Fukuzawa et al.
2006). These observations could be explained by
formation of a variety of radicals during the reduction in peroxide groups which exhibit the ability
to further oxidize additional amounts of Fe2+.
Moreover, Wolff (1994) and Gay et al. (1999) also
cautioned that different peroxides produce significantly different amounts of Fe3+ and therefore require
determination of compound-specific molar absorption coefficients. It was further stressed that the
common assumption of same molar absorption
coefficients for peroxides as varied as H2O2, lipids,
cumene, tert-butyl, proteins, and serum could be a
source of error in the analysis.
In consideration of these facts, a standard calibration curve was established with both the HPO mix
and H2O2 that showed linear curve fit (Fig. 1 and

DISCUSSION

Hydroperoxide determination is gaining considerable importance due to its central role in cell signaling and generation of defense compounds besides
being an indicator of oxidative stress. The LHPOs
influence on cellular processes is often arbitrated
by their self-perpetuation and amelioration by

TABLE 3. Lipid hydroperoxide contents in Ulva lactuca cultured in artificial seawater medium (ASW) supplemented with
different nutrients (N, P, and NP) and ASW alone as control. Values are given as mean SD (n = 3).
FOX assay

Samples

ASW
ASW + N
ASW + P
ASW + NP

HPO mix. calibration

(lg
g1 f.w.)

44 10a
27 9b
32 2ab
26 2b

TBARS assay

H2O2 equivalents
(lmol
g1 f.w.)

1.83
1.15
1.32
1.07

0.4a
0.4b
0.1ab
0.1b

[lg
g1 f.w.]

[62
[39
[45
[36

13a]
13b]
3.5ab]
2.3b]

LHPOs/TL

0.0032
0.0016
0.0011
0.0013

0.001a
0.002b
0.001b
0.0003b

(lmol
g1 f.w.)

0.04
0.03
0.05
0.02

0.002b
0.003bc
0.001a
0.006c

[lg
g1 f.w.]

[1.26
[1.14
[1.65
[0.69

0.08b]
0.09bc]
0.04a]
0.22c]

Values in a column are significantly different at P  0.05.

ac

TABLE 4. Polyunsaturated fatty acid composition and specific lipid peroxidazibility of Ulva lactuca cultured with different
nutrient sources, data expressed as mean SD (n = 3).
Parameters

PUFAs contents (in lg
mg1 lipid)

C18:2 (n6)
C18:3 (n6)
C18:3 (n3)
C18:4 (n3)
C20:3 (n6)
C20:4 (n6)
C20:5 (n3)
C22:6 (n3)
Double bond index (DBI) ()A
Specific lipid peroxidazibility (SLP) (lg
1)

ASW

2.44
0.05
3.13
1.28
0.07
0.10
0.11
0.46
1.0
42.3

0.04a
0.02
0.23
0.34
0.01
0.01
0.01
0.1
0.08
9.25a

ASW + N

1.04
0.04
2.33
0.71
0.04
0.06
0.07
0.16
1.23
22.4

0.58b
0.03
1.18
0.4
0.06
0.08
0.09
0.2
0.42
7.5b

ASW + P

0.63
0.02
1.3
0.52
0.02
0.09
0.08
0.13
1.2
26.4

0.16b
0.01
0.34
0.15
0.01
0.03
0.02
0.06
0.3
2.1b

ASW + NP

1.06
0.04
3.32
0.78
0.06
0.08
0.06
0.17
1.6
16.6

A Double bond index = [2 9 (%C18:2) + 3 9 (C18:3) + 3 9 (C20:3) + 4 9 (C20:4) + 5 9 (C20:5) + 6 9 (C22:6)]/100.

ab
Values in a row are significantly different at P  0.05.

0.67b
0.02
2.9
0.53
0.01
0.02
0.01
0.07
0.37
1.05b

LIPID HYDROPEROXIDES IN MACROALGAE

Table S1). However, calculations were made on the

basis of the calibration curve of HPO mix on the
presumption that macroalgae contain appreciable
amounts of both the C18 and C20 PUFAs as
revealed in our present (Tables 2 and 4) and earlier
studies (Kumari et al. 2010, 2011, Kumar et al.
2011), and thereby estimation of hydroperoxides
merely based on H2O2 or linoleic acid hydroperoxide could be misleading. This proposition was
confirmed by 1.4-times higher values obtained for
all the macroalgal samples expressed in terms of
H2O2 equivalents than those obtained from LHPO
mix calibration curve (Tables 1 and 3). These
higher estimates may be because of the higher reactivity of H2O2 for the FOX reagent (Jiang et al.
1992, Nourooz-Zadeh et al. 1994, 1995, DeLong
et al. 2002). Our findings indicate that the expression of LHPO values in the form of H2O2 equivalents give higher estimates, thus confirming the
proposition of Wolff (1994) and Gay et al. (1999)
that using the same molar absorption coefficient for
different types of hydroperoxides (H2O2, lipid, cumene, and others) could give erroneous results.
Besides this, Vega et al. (2005) advocated for establishing a proper calibration curve based on same
components that are present in the reaction medium to administer the presence of nonperoxidized
lipids on the sensitivity of the assay. Thus, the calibration curves of lipid extracts of U. lactuca, G. corticata,
and S. tenerrimum belonging to green, red, and
brown macroalgae, respectively, were studied, and a
linear relationship was found between the amount
of tissue extract added to the reaction medium and
absorbance, in agreement with the earlier report of
Hermes-Lima et al. (1995) for animal tissue extracts
(Fig. S1 and Table S2, see Supporting Information).
It is well known that ROS are photochemically
produced in seawater by the absorption of solar
radiation, especially UVR (290400 nm), by dissolved organic matter. H2O2 has the highest shelf
life and steady state concentrations (107 M) in seawater and can readily pass through biological membranes (Mopper and Kieber 2000). These ROS
could pose deleterious effects on marine algae by
affecting cell membranes and potentially inhibiting
photosynthesis. Thus, algae require robust meta-

1369

bolic adjustments and antioxidant defenses to

maintain these ROS at low levels. The low
incipient levels of LHPOs detected in the investigated macroalgae indicated the presence of a
similar strong antioxidant defense system (consisting of carotenoids, polyphenols, glutathione, and
antioxidant enzymes SOD, POD, CAT) that
maintains such low concentrations of LHPOs
despite being exposed to diverse adverse environmental conditions, such as extremes of light, temperature, salinity, numerous endo- and epiphytic
bacteria, fungi, and microalgae in the sea. Zubia
et al. (2007) also found an enhanced protective
capacity in tropical marine macrophytes against
ROS products that could cause oxidation of PUFAs
in cell membrane, leading to lipid peroxidation and
then to cell damage. Moreover, the LHPO contents
in green macroalgae were lower than that reported
in C. reinhardtii by Griffiths et al. (2000), but the
LHPOs contribution to the total fatty acid fraction
(1%2%) was relatively higher in this study than
in C. reinhardtii (0.8%) and other tissue extracts
(0.6%1.7%). This may be due to the higher ability
of the Chlamydomonas sp. to accumulate lipids. Similarly, DeLong et al. (2002) reported higher LHPO
contents for plant tissue extracts of avocado, pear,
potato, red cabbage, red pepper, and spinach than
the macroalgal samples investigated in this study.
A novel approach of specific lipid peroxidazibility
(SLP) was introduced by Fakas et al. (2008) for evaluating the susceptibility of lipids to peroxidation for
studying lipid peroxidation in an oleaginous fungus
Cunninghamella echinulata. The SLP values reported
for different macroalgae in this study, which were in
the range 4.6116.1 lg
1, were significantly
lower than those recorded for different lipid classes
for C. echinulata. Girotti (1998) suggested that lipid
peroxidazibility increases with the PUFAs contents
and DBI of the lipids. In contrast, no correlation
was found between PUFAs and LHPOs, as evident
from the PCA analysis in this study, instead, a negative correlation was observed for SLP and DBI
(Fig. 2). This is in agreement with the findings of
Fakas et al. (2008), wherein reported that PUFA
peroxidazibility depends on the nature of the lipid
fraction in which they are located than the PUFA

FIG. 1. Standard calibration curves of (A) HPO HPLC Mix and (B) H2O2 for FOX2 assay.

1370

PU J A K U M A R I E T A L .

contents alone. The PUFAs esterified in polar lipids

especially phospholipids were more susceptible to
peroxidation than those esterified in glycerol
because phospholipids are membrane bound and
thus are more available to the radicals formed in
the membrane lipids as compared to neutral lipids
that reside in cytoplasmic lipid bodies. Although we
did not study the detailed LHPO contents of different lipid classes (phospholipids, glycolipids, and
neutral lipids) of macroalgae, the LHPOs determined in the crude lipid extract corroborates well
with the findings of Fakas et al. (2008) that lipid
peroxidation was found to have no correlation with
PUFA contents (Fig. 2).
PCA analysis further revealed a species-specific
distribution of LHPOs with all Sargassum species
forming a separate group from rest of the species,
while no distinct distribution patterns were exhibited among the green, red, and brown macroalgae.
This result is in contrast with their fatty acid distribution matrices, where green and red algae are
widely separated from each other, brown algae
being sandwiched between the two groups (Kumari
et al. 2010). This result may be due to the fact that
this study was confined to a few genera including
Ulva, Codium, Chamaedoris, Gracilaria, Sarconema,
Sargassum, Dictyota, Padina, and Spatoglossum. Possibly
a more extensive analysis of macroalgal samples
could have provided more clear trends, if any exist
regarding distribution of their hydroperoxides and
peroxidation susceptibilities.
Algal thalli exhibit various biochemical adaptations
to cope with the fluctuating nutrients availability by
altering pigments, lipid composition, FAs, expression
of enzymes, and nutrient uptake (Grarcia-Sanchez
et al. 1996, Ale et al. 2010). The higher LHPO
contents and TBARS-MDA complex recorded for
U. lactuca cultured in ASW as compared to those

supplemented with N and P signifies a state of

induced oxidative stress as a result of nutrient
imbalance. Li et al. (2005) reported increased
LHPOs (MDA) levels in the microalga Pavlova viridis
under nitrate starvation. Yu and Yang (2008) also
reported an increase in hydroperoxide, superoxide,
and peroxide contents and the interplay of SOD,
POD, CAT in Gracilariopsis lemaneiformis to concentration changes of N and P. Similar increased hydroperoxides have been reported in higher plants for
mulberry grown under nitrogen, phosphorus, potassium, and boron deficiencies (Tewari et al. 2007,
2009), resulting in a state of nutrient limitation
induced oxidative stress by increasing ROS generation and concomitant up-regulation of antioxidant
enzymes to minimize the stress to a certain extent.
The comparative analysis of the FOX2 assay and
the TBARS method of lipid peroxidation in different macroalgal samples including U. lactuca cultured with different nutrients revealed that the
FOX2 assay gave  20-fold higher contents for
LHPOs as compared to TBARS. This discrepancy
may be attributed to the inability of fatty acids containing two double (=) bonds (such as linoleic acid
to form MDA) which is mainly formed from fatty
acids containing 3 or more double bonds. As macroalgae irrespective of their groups (including
U. lactuca) contains appreciable amounts of linoleic
acid (Tables 2 and 4), it is therefore not surprising
that the TBARS assay gave lower values. Jentzsch
et al. (1996) have clearly described the nonspecificity of the TBARS assay, stating that chromogens
could be formed with many aldehydes other than
MDA, carbohydrates, and amino acids, and that
these problems get even worse when applied to biological samples. Hermes-Lima et al. (1995) also
reported that the FOX assay gave 69-fold higher
values for lipid peroxidation than TBARS for Cu+

FIG. 2. Principal component analysis of 25 different macroalgal samples; bi-plot of LHPO, LHPO/TL, LHPO/TFA, SLP, PUFAs, and
DBI with first two principal components.

LIPID HYDROPEROXIDES IN MACROALGAE

induced peroxidation in liposomes. They further

estimated that the formation of Fe (III) xylenol
orange complex during the FOX assay is a 1020fold amplification of the original levels of hydroperoxides in tissue extracts. This amplification
property makes it possible to perform sensitive
comparative studies of the extent of lipid peroxidation in vivo and contributes to its high sensitivity
by detecting even very low levels of LHPOs. Thus,
this study reveals that TBARS does not seem to be
an appropriate method for determining the lipid
peroxidation in macroalgae.
In conclusion, the FOX assay permits the quantification of low concentrations of LHPOs, despite the
presence of high background levels of nonperoxidized fatty acids, and this assay has the potential to
be a rapid, sensitive, and inexpensive method for
detecting incipient lipid peroxidation in macroalgal
tissues. The interplay of strong antioxidant defense
systems maintains the low levels of LHPOs in macroalgae under different biotic, abiotic, and nutrient
stresses. The susceptibility of macroalgal lipid peroxidation is independent of both the PUFAs content
and their degree of unsaturation (DBI). Furthermore, precautions should be exercised to perform
all the analyses with the same lot of xylenol
orange to minimize the source of error and to
improve the reproducibility and reliability of the
assay.
We thank Chief Conservator of Forest and Wildlife, Jamnagar
and Ministry of Forest and Environment, Govt. of Gujarat, for
permitting us to collect algal samples from Kalubhar Island
(off-Vadinar Coast), Marine National Park, Gujarat. We
express our sincere gratitude to the Head, Analytical Sciences
for permitting us to carry out some analytical work reported
in this study. Prof. Michael Wynne, University of Michigan,
USA is also gratefully acknowledged for his suggestions on
the taxonomic aspects and reviewing of revised manuscript.
PK and RPS acknowledge CSIR, New Delhi, for their senior
research fellowships (CSIR-SRFs).
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Supporting Information
Additional Supporting Information may be
found in the online version of this article at the
publishers web site:
Table S1. Comparison of sigmoidal curve fit
and linear curve fit model for calibration curves
of standards, Hydroperoxy HPLC Mix and H2O2
(at P  0.05), obtained using OriginPro 8.5.1.
Table S2. Comparison of sigmoidal curve fit
and linear curve fit model for calibration curves
of lipid hydroperoxides of algal lipid extracts of
Ulva lactuca, Gracilaria corticata, and Sargassum
tenerrimum (at P  0.05), obtained using OriginPro 8.5.1.
Figure S1. Calibration curves of lipid hydroperoxides of algal lipid extracts of Ulva lactuca,
Gracilaria corticata, and Sargassum tenerrimum,
where y1, y2, and y3 are their respective linear fit
curves.