Biosci. Biotechnol. Biochem., 70 (11), 2598–2603, 2006
Oxylipin Metabolism in Soybean Seeds Containing Different Sets
of Lipoxygenase Isozymes after Homogenization
Zinna Marie P ULVERA,1 Keisuke K ITAMURA,2 Makita H AJIKA,3
Kazuko S HIMADA,4 and Kenji M ATSUI1; y
1
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
Department of Plant Breeding Science, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
3
National Institute of Crop Science, Tsukuba 305-8518, Japan
4
Department of Human Nutrition, Faculty of Human Life Science, Yamaguchi Prefectural University,
Yamaguchi 753-8502, Japan
2
[doi:10.1271/bbb.60121]
The oxylipin metabolism was analyzed in soybean
homogenates containing different sets of lipoxygenase
isozymes (L-1, -2, and -3); namely, Suzuyutaka (containing L-1, -2, and -3), Yumeyutaka (containing only
L-1), Kanto102 (containing L-2), Kyushu119 (containing
L-3), and Ichihime (lacking all three isozymes). The
amount of oxidized fatty acids in the esterified form was
higher than that in the free form with every cultivar.
Kanto102 formed the highest amount of oxidized lipids,
and Yumeyutaka and Ichihime formed the lowest. With
Kanto102 and Kyushu119, high amounts of keto fatty
acids were formed, while they were undetectable with
Yumeyutaka and Ichihime. Due to the lack of lipoxygenases in Ichihime, an accumulation of free fatty acids
was expected; however, their amount in Yumeyutaka
was significantly lower than was expected. It is suggested that a pathway existed to form C6-volatiles through
hydroperoxides in the esterified form.
Key words:
lipoxygenase; oxylipin metabolism; lipid
hydroperoxide; soybean; Glycin max
Lipoxygenase is an enzyme that catalyzes the insertion of molecular oxygen onto fatty acids containing
(Z,Z)-1,4-pentadiene moiety, such as linoleic and linolenic acids in plants. The hydroperoxides thus formed
are further metabolized to form a wide variety of
compounds which are cumulatively called oxylipins
(Fig. 1).1) Short-chain aldehydes are formed from the
hydroperoxides by fatty acid hydroperoxide lyase
(HPL). These aldehydes with their alcohols and acetates
consist of a subgroup of oxylipins and are called green
leaf volatiles (GLVs).2) GLVs are important flavor
y
constituents in such food materials as tomatoes,3) while
in other food materials, especially soybeans, GLVs
cause an unpleasant off-flavor.4) The beany flavor of
soybean is due to n-hexanal and n-hexan-1-ol and is
believed to be one of the major factors limiting their use
in foods. It has been shown that soybean seeds have
three isozymes of lipoxygenases, i.e., lipoxygenase-1,
-2, and -3 (L-1, -2, and -3), which exhibit different
substrate/product specificity and kinetic behavior.5)
Intense breeding has succeeded in producing soybeans
lacking one or two, or even all the three lipoxygenases.6,7) Volatile analyses using these genetic resources
have enabled Matoba et al.8) to find that L-2 mainly
contributed to the formation of GLVs. Extensive volatile
analyses performed by Kobayashi et al.9) have also
shown the importance of L-2 in forming GLVs, and
demonstrated the superiority of a new cultivar that
lacked all three lipoxygenases as a material for soymilk
production.
Lipoxygenase products, namely, oxidized metabolites
of fatty acids, can lead to a loss of nutritional value due
to destruction of vitamins, amino acids and proteins.10)
Nishida et al.11) have reported that the amounts of lipid
peroxides rapidly increased after the homogenization of
soybean seeds and that lipoxygenase-lacking soybean
varieties formed a lower amount of lipid peroxides.
However, little is known about the composition and
content of lipid peroxides and their derivatives in a
soybean homogenate whose biological activities could
depend on its structure. We analyzed in this study the
composition of lipid peroxides formed after the homogenization of soybean seeds having different sets of
lipoxygenase isozymes.
To whom correspondence should be addressed. Tel: +81-83-933-5850; Fax: +81-83-933-5820; E-mail: matsui@yamaguchi-u.ac.jp
Abbreviations: GLV, green leaf volatile; HPL, hydroperoxide lyase; H(P)OD, hydro(pero)xyoctadecadienoic acid; KOD, ketooctadecadienoic acid;
L-1 (-2, -3), soybean lipoxygenase-1 (-2, -3); L123 , Suzuyutaka; L1 , Yumeyutaka; L2 , Kanto102; L3 , Kyushu119; L0 , Ichihime
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Received March 8, 2006; Accepted June 28, 2006; Online Publication, November 7, 2006
Oxylipin Metabolism in Soybean
2599
OH
+
1-Octen-3-ol
COOR
OHC
10-Oxo-(E)-8-decenoic acid
Lyase (?)
COOR
Linoleic acid 10-hydroperoxide
HOO
Oxygenase (?)
O2
COOR
Linoleic acid
9-Lipoxygenase
O2
13-Lipoxygenase
HOO
COOR
13(Z,E)HPOD
9(E,Z)HPOD
COOR
Fatty acid 13hydroperoxide lyase
CHO + OHC
n-Hexanal
O
O 13(Z,E)KOD
COOR
9(E,Z)KOD
COOR
12-Oxo-(Z)-9-dodecenoic acid
Alcohol
dehydrogenase
CH2OH
n-Hexan-1-ol
Fig. 1. Oxygenation of Linoleic Acid and Its Ester in Soybean Homogenate.
Linoleic acid is oxygenized to form 13- or 9-HPOD. KODs are formed through secondary reaction of lipoxygenases from HPODs. n-Hexanal
is formed by fatty acid 13-hydroperoxide lyase, and further converted into n-hexan-1-ol by alcohol dehydrogenase. 1-Octen-3-ol may be formed
through oxygenation and subsequent cleavage of linoleic acid; however, the enzymes involved in the formation of 1-octen-3-ol have not been
elucidated. The compound names are for those having free carboxylic acid (R ¼ H). It might be possible for linoleic acid esterified to
triacylglycerols to enter into the oxygenation pathway shown here. In this case, R could be diacylglycerol. 13(Z,E)HPOD, 13-hydroperoxy(9Z,11E)-octadecadienoic acid; 13(Z,E)KOD, 13-oxo-(9Z,11E)-octadecadienoic acid; 9(E,Z)HPOD, 9-hydroperoxy-(10E,12Z)-octadecadienoic
acid, 9(E,Z)KOD, 9-oxo-(10E,12Z)-octadecadienoic acid.
It is widely accepted that GLVs are formed from free
fatty acids,2) largely because most plant lipoxygenases
prefer free fatty acids. However, it has been also
reported that some lipoxygenases can act on esterified
acyl groups when the lipid substrates are appropriately
prepared.12,13) Analyses of the composition and quantity
of oxylipin metabolites formed after the homogenization
of soybean seeds also suggested that esterified fatty
acids in the soybean seed homogenate constituted a
good substrate for lipoxygenases. A comparison of the
amounts of oxylipin metabolites in the soybean varieties
suggested for the first time that a pathway to form C6volatiles without releasing free fatty acids was operating
in each soybean homogenate.
Materials and Methods
Materials. Soybeans [Glycine max (L.) Merr.] were
harvested at the Kyushu National Agricultural Experiment Station (Kumamoto, Japan) in 2003–2004, and
stored at 5–7 C until their use. Normal soybean,
Suzuyutaka, contains L-1, -2, and -3 (abbreviated as
L123 based on the remaining isozymes). Yumeyutaka
(L1 , containing only L-1), Kanto102 (L2 , containing
only L-2), and Kyushu119 (L3 , containing only L-3) are
varieties containing only one lipoxygenase isozyme.
Ichihime is a variety lacking all the three lipoxygenase
isozymes,6) and is designated as L0 in this paper.
Linoleic acid (99% pure) and eicosadienoic acid
(98%) were purchased from Sigma (St. Louis, MO,
USA). Linoleic acid 13-hydroperoxide (13-HPOD) and
eicosadienoic acid 15-hydroperoxide (15-HPED) were
prepared by using soybean L-1 as previously described.14) The corresponding hydroxides were prepared by
reducing them with triphenylphosphine. 13- and 9-oxo
linoleic acids (13- and 9-KOD) were purchased from
Cayman Chemical (Ann Arbor, MI, USA). n-Hexanal,
n-hexan-1-ol, 1-octen-3-ol, and n-pentan-1-ol were
obtained from Wako Pure Chemicals (Osaka, Japan).
All other chemicals were of analytical grade.
Analyses of volatiles by SPME. One soybean seed
(0.2–0.3 g) was placed in a glass vial and soaked
overnight in 3 ml of water. After homogenizing for
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HOO
COOR
Z. M. P ULVERA et al.
Analyses of hydroperoxy and hydroxy fatty acids.
Soybean seeds (2.5 g) were soaked overnight in 15 ml of
water, homogenized with a Polytron homogenizer (PTA
10S, Kinematica) and incubated at room temperature
for 1 hr. After adding 300 ml of 3.6 mM 15-HPED as an
internal standard, the homogenate was slowly acidified
to pH 4 with HClO4 (60%), and the acidic compounds
were extracted with 60 ml of chloroform:methanol
(50:50, v/v). The resulting aqueous phase was further
extracted with 30 ml of chloroform, and both the organic
phases were combined and dried over anhydrous sodium
sulfate. To reduce the hydroperoxy group, 2.5 ml of 0.1 M
triphenylphosphine (in ether) was added, and the solvent
was later removed under vacuum. The residue was
hydrolyzed by heating at 80 C for 15 min in 2.5 ml of
3.5N NaOH containing 2.5 ml of ethanol. After cooling,
the mixture was acidified to pH 4 with 3.5N HClO4 , and
the hydrolyzed products were extracted with 1.5 ml of
hexane:ether (70:30, v/v). An aliquot of the extract
(2 ml) was analyzed by straight-phase HPLC, using a
Zorbax-rx-SIL column (4:6 250 mm, Agilent, USA)
with a solvent of hexane:ether:acetic acid (70:30:0.25,
v/v/v) at a flow rate of 1.0 ml/min. A photodiode array
(SPD-M10AVP, Shimadzu) was used for detection.
Almost all the positional and geometric isomers of the
hydroxides and ketones of linoleic acids could be
separated under the experimental conditions employed
here. 13-Oxo-(E,E)- and 9-oxo-(E,Z)-octadecadienoic
acids could not be separated, so, the sum of these two
isomers is shown. There was one unidentified oxygenated compound at a retention time of 25 min. This
showed max at 234 nm, so, it was thought to be a
compound containing a 1-hydro(pero)xy-2,4-diene moiety. This compound is subsequently referred to as
unknown 1. No oxidized metabolites of the other fatty
acids, such as those derived from linolenic acid, could
be detected in the homogenate of any of the soybean
varieties used in this study. When the hydrolysis step
was omitted from the procedure, only the oxidized fatty
acids in their free form could be detected. The quantity
of the esterified form was estimated from the difference
in the amount of the total lipid peroxides and that of the
free type. Each hydroxy fatty acid was quantified by
using a molecular coefficient of 23000. 19600 and 22300
were respectively used for 9- and 13-KOD.
Determination of the free fatty acids. The acidic
fraction was extracted from a soybean homogenate as
just described but with margaric acid (600 ml of 3.69 mM
in ethanol) used as the internal standard. A portion of
the extract was spotted on a TLC plate (silica gel 60
F254, Merck) and developed with hexane:ether:acetic
acid (80:50:1, v/v/v). The spot corresponding to the free
fatty acids was scraped off, and the free fatty acids were
extracted with ether, before being esterified with
ethereal diazomethane. A GC–MS analysis was conducted as already described but with a column temperature from 80 C to 190 C at a rate of 10 C/min, and
then from 190 C to 230 C at 10 C/min. Each fatty
acid methyl ester was identified by comparing its
retention time and MS profile with those of an authentic
specimen.
Results and Discussion
Formation of volatiles
The volatile analyses by SPME showed n-hexanal,
n-hexan-1-ol, and 1-octen-3-ol to be the major volatile
compounds in the soybean seed homogenates (Fig. 2).
With L123 , the amounts of these three volatile compounds were almost same as those reported by
Kobayashi et al.9) L2 formed higher amounts of n-
n-hexanal
5.0
1-octen-3-ol
n-hexan-1-ol
4.0
3.0
2.0
1.0
0
L123
L2
L3
L1
Soybean genotype
L0
Fig. 2. Amounts of Volatiles Emitted from Crude Homogenates
Prepared from the Seeds of Suzuyutaka (L123 ), Yumeyutaka (L1 ),
Kanto102 (L2 ), Kyushu119 (L3 ) and Ichihime (L0 ).
Each soybean seed homogenate was incubated at 25 C for
30 min, and the amounts of n-hexanal (white bar), n-hexan-1-ol
(shaded bar), and 1-octen-3-ol (striped bar) formed were determined
by SPME-GC. Error bars show SD (n ¼ 3).
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3 min, the homogenate was incubated at room temperature for 30 min. Saturated CaCl2 (3 ml) was added to
stop the enzymatic reaction, and 10 ml of 100 mM npentan-1-ol (in ethanol) as an internal standard and 6 ml
of 0.1 M nordihydroguaiaretic acid (in ethanol) as an
antioxidant were then added. The vial was sealed with a
cap fitted with a Teflon septum and the content was
thoroughly mixed. The sample was heated at 80 C and,
after equilibrating for 2 min, SPME fiber (diviniylbenzene/carboxen/polydimethylsiloxane, Supelco) was exposed to the headspace gas for 30 min at 80 C. The
volatiles were desorbed at 240 C for 1 min in the
injection port of GC-MS apparatus (QP5050, Shimadzu)
equipped with a DB-WAX column (60 m 0:25 mm,
0.25 mm, Agilent). The initial oven temperature was
50 C, this being increased at 10 C/min to 180 C. The
mass spectrometer was operated in the electron ionization mode at 70 eV. A component was identified by
comparing its retention time and mass spectra with those
of an authentic compound.
Amount ( µ mol/g of seed)
2600
Oxylipin Metabolism in Soybean
Accumulation of oxidized fatty acids
It has been reported that the amounts of oxidized
lipids were low in intact soybean seeds, but that they
increased after homogenization.11) In L123 , the most
abundant oxidized fatty acid was esterified 13-H(P)OD,
followed by free 13-H(P)OD, the former being three
times more abundant (Fig. 3). Its (Z,E)-isomer was
predominant in each form. 9-H(P)OD could also be
detected in both the esterified and free forms. The
amounts of its (E,Z)- and (E,E)-isomers were almost
same as each other. 9- and 13-KODs were also formed.
Although L2 lacked both L-1 and L-3, a higher
formation of oxidized fatty acids than in L123 could be
found. In particular, the amount of 13-H(P)OD in either
the esterifed or free form was two times higher than that
formed with L123 . Again, its (Z,E)-isomer was predominant, while 9-H(P)OD was composed of almost equal
amounts of the (E,Z)- and (E,E)-isomers. The amounts
of 9- and 13-KODs in the esterified form were high,
while, those in the free form were low. With L3 , almost
the same amounts of 9- and 13-H(P)OD were formed.
The amounts of KODs were high, especially in the
esterified form. The enzyme reaction after homogenizing the L1 seeds resulted in the preferential formation of
13-H(P)OD in the esterified form, although the amount
was lower than that found with L123 or L2 . The
formation of KODs could not be detected in either the
esterified or free form. Even H(P)OD formation in the
free form could not be detected with L1 . With L0 , 13H(P)OD was the major oxidized fatty acid, this being
followed by 9-H(P)OD, and both of these were solely
found in the esterified form. Their amounts were still
Oxidized fatty acids (nmol/g of seed)
1600
13(Z,E)HOD
1200
13(E,E)HOD
9(E,Z)HOD
800
9(E,E)HOD
Unknown 1
13(Z,E)KOD
13(E,E)KOD +
9(E,Z)KOD
400
9(E,E)KOD
0
Free form
400
0
nd
L123
L2
L3
L1
nd
L0
Soybean genotype
Fig. 3. Amounts of Oxidized Fatty Acids in the Esterified and Free
Forms Formed in the Crude Homogenates Prepared from Seeds of
Suzuyutaka (L123 ), Yumeyutaka (L1 ), Kanto102 (L2 ), Kyushu119
(L3 ) and Ichihime (L0 ).
13(Z,E)HOD,
13-hydroxy-(9Z,11E)-octadecadienoic
acid;
13(E,E)HOD,
13-hydroxy-(9E,11E)-octadecadienoic
acid;
9(E,Z)HOD, 9-hydroxy-(10E,12Z)-octadecadienoic acid; 9(E,E)HOD, 9-hydroxy-(10E,12E)-octadecadienoic acid; 13(Z,E)KOD,
13-oxo-(9Z,11E)-octadecadienoic acid; 13(E,E)KOD, 13-oxo(9E,11E)-octadecadienoic acid; 9(E,Z)KOD, 9-oxo-(10E,12Z)-octadecadienoic acid; 9(E,E)KOD, 9-oxo-(10E,12E)-octadecadienoic
acid. Error bars show SD (n ¼ 3).
lower than those found with L1 homogenate. No KODs
could be found. It must be noted that the amount of lipid
peroxides accumulated in each soybean genotype used
in this study was roughly one-sixth of the amount of C6volatiles.
As expected, the amounts and compositions of
oxidized lipids formed in the soybean homogenates
varied extensively according to the lipoxygenase isozymes the seeds possessed. It should be noted that the
esterified form of oxidized fatty acids was always
predominant with any of the soybean varieties. Esterified hydroperoxides might have been mostly formed
by L-2 and -3, as Zhuang et al.16) have reported that
they could act on esterified fatty acids, while L-1 hardly
could. However, even with L1 , the formation of
esterified hydroperoxides was evident, probably because
the lipids were appropriately emulsified in the homogenate to be a good substrate for L-1.12) Another notable
result here is that L2 and L3 formed high amounts of
KODs, especially in the esterified form. KODs might be
formed via the secondary reaction of lipoxygenases.17)
This is the first report on the formation of KODs in the
esterified form.
Accumulation of free fatty acids
The formation of free fatty acids after homogenization was analyzed with the representative varieties of
soybean, L123 and L0 . The most abundant free fatty acid
formed with L123 after homogenization was palmitic
acid. Linoleic and stearic acids followed; however, the
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hexanal and n-hexan-1-ol, while L3 formed only one
third and one fifth of the respective amounts of nhexanal and n-hexan-1-ol that were found in the L123
homogenate. L1 formed still lower amounts of n-hexanal
and n-hexan-1-ol. The amounts of these two C6 volatiles
in the L0 homogenate were almost the same as those
found in the L1 homogenate, even though L0 lacks all
the three seed lipoxygenases. The amount of 1-octen-3ol was constant among the soybeans used here, as
reported previously.9) When the seeds were homogenized with a saturated CaCl2 solution in order to
immediately inactivate most enzymes, hardly any of
the C6 compounds and 1-octen-3-ol were formed,
indicating that they were enzymatically formed after
homogenization. When L123 seeds were homogenized in
the presence of the lipoxygenase inhibitor, nordihydroguaiaretic acid, there was little formation of C6compounds, although the amount of 1-octen-3-ol formed
was little affected by the inhibitor (data not shown). It is
therefore suggested that 1-octen-3-ol was formed independently with the typical type of lipoxygenase, even
though the involvement of the 10-hydroperoxide of
linoleic acid, which might be formed by an oxygenase
reaction has been reported in the formation of 1-octen-3ol.15)
2601
Z. M. P ULVERA et al.
Amount (nmol/g of seed)
2602
L123
1200
L0
1000
800
600
400
200
0
C14:0 C16:0 C18:0 C18:1 C18:2 C18:3
Fatty acid
Acknowledgments
formation of free oleic acid, which is the second most
abundant acyl group found in the lipids of soybean
seeds,18) was relatively low (Fig. 4), suggesting uneven
hydrolysis of the acyl groups and/or uneven metabolism
of the free fatty acids. When L0 seeds were homogenized, the amounts of palmitic, stearic, and linoleic acids
hydrolyzed were higher than those with L123 seeds. The
accumulation of free linoleic acid was most evident, the
difference in the amount of free linoleic acid with L0 and
L123 being about 0.7 mmol/g of seed. It should be noted
that free palmitic and stearic acids were also accumulated in L0 , even though these fatty acids were not
substrates for lipoxygenases.
It is believed that C6-volatiles are formed through the
oxygenation of free fatty acid to yield corresponding
hydroperoxides.2) The amounts of free fatty acids and
their hydroperoxides in most intact plant tissues are
generally low, so lipid hydrolysis must be the first
commitment step to form C6-volatiles. It could be
expected that free fatty acids would accumulate in the
L0 homogenate because of the lack of all three lipoxygenases. However, the difference in the amounts
found here (0.7 mmol/g of seed) is not comparable with
the difference in the amounts of C6-volatiles between
L123 and L0 (3.5 mmol/g of seed). This result suggests
that the C6-volatiles might be formed at least partly
without lipid hydrolysis to form unoxidized free fatty
acids. Soybean seed lipoxygenases can act on esterified
fatty acids.13,14) This study has also shown that hydroperoxides could be found in their esterified forms,
suggesting that direct oxidation of esterified lipids might
account for the formation of C6-volatiles. HPL may be
able to act directly on the esterified hydroperoxides, or
indirectly after hydrolysis of the esters to form fatty acid
hydroperoxides. Arabidopsis HPL can act on the methyl
ester of 13-HPOD,19) which suggests that the terminal
free carboxyl group is not essential for HPL catalysis.
Taken together, it can be assumed that this ‘alternate’
pathway to form C6-aldehydes in a soybean seed
Z.M.P. is supported by a scholarship for foreign
students from the Ministry of Education, Culture, Sports,
Science and Technology of Japan. This study was partly
supported by Grant-in-Aid (15380009 to KK) from
Japan Society for the Promotion of Science.
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