Characterization and quantification of free and esterified 9- and 13-hydroxyoctadecadienoic acids (HODE) in barley, germinating barley, and finished malt

Article abstract of DOI:10.1021/jf048490s

The analysis of (R)-9- and (S)-9-hydroxy-10E,12Z-octadecadienoic acid as well as (R)-13- and (S)-13-hydroxy-9Z,11E-octadecadienoic acid (HODE) as free acids, esterified in triacylglycerols (storage lipids), and esterified in polar lipids (phospholipids, glycolipids, etc.) in barley, germinating barley, and finished malt was performed using [13-¹⁸O₁]-(S)-13-HODE isotope dilution assays with GC-MS and straight- and chiral-phase HPLC. 9- and 13-HODE occur approximately racemically in barley, indicating an autoxidation. The enantiomeric excesses increase to 78% S for free 9-HODE and to 58% S for free 13-HODE in germinating barley as a result of lipoxygenase-2 (LOX-2) catalysis, but free HODEs are at low concentration. More than 90% of HODEs in barley and malt are esterified. In the storage lipids of green malt 53 mg/kg 9-HODE and 147 mg/kg 13-HODE were detected. This ratio of 30:70 reflects the regioselectivity of the LOX-2 enzyme in malt. In the polar lipids 45 mg/kg 9-HODE and 44 mg/kg 13-HODE were characterized. The latter indicate a hitherto unknown 9-lipoxygenase activity with polar lipids as substrates. During kilning the contents of most HODEs decreased significantly due to chemical and enzymatic degradation, whereas polar-esterified (R)-13-HODE increased (43%) in the finished malt.

Full text of DOI:10.1021/jf048490s

1556 J. Agric. Food Chem. 2005, 53, 1556−1562
Characterization and Quantification of Free and Esterified 9-
and 13-Hydroxyoctadecadienoic Acids (HODE) in Barley,
Germinating Barley, and Finished Malt
HOLGER HU¨ BKE, LEIF-ALEXANDER GARBE,* AND ROLAND TRESSL
Institut fu¨r Biotechnologie, Molekularanalytik, Technische Universita¨t Berlin,
Seestrasse 13, D-13353 Berlin, Germany
The analysis of (R)-9- and (S)-9-hydroxy-10E,12Z-octadecadienoic acid as well as (R)-13- and (S)-
13-hydroxy-9Z,11E-octadecadienoic acid (HODE) as free acids, esterified in triacylglycerols (storage
lipids), and esterified in polar lipids (phospholipids, glycolipids, etc.) in barley, germinating barley,
and finished malt was performed using [13-18O1]-(S)-13-HODE isotope dilution assays with GC-MS
and straight- and chiral-phase HPLC. 9- and 13- HODE occur approximately racemically in barley,
indicating an autoxidation. The enantiomeric excesses increase to 78% S for free 9-HODE and to
58% S for free 13-HODE in germinating barley as a result of lipoxygenase-2 (LOX-2) catalysis, but
free HODEs are at low concentration. More than 90% of HODEs in barley and malt are esterified. In
the storage lipids of green malt 53 mg/kg 9-HODE and 147 mg/kg 13-HODE were detected. This
ratio of 30:70 reflects the regioselectivity of the LOX-2 enzyme in malt. In the polar lipids 45 mg/kg
9-HODE and 44 mg/kg 13-HODE were characterized. The latter indicate a hitherto unknown
9-lipoxygenase activity with polar lipids as substrates. During kilning the contents of most HODEs
decreased significantly due to chemical and enzymatic degradation, whereas polar-esterified (R)-
13-HODE increased (43%) in the finished malt.
KEYWORDS: Lipoxygenase; barley; green malt; malt; analysis; HODE; polar lipids; enantiomers
INTRODUCTION
synthases and epoxy alcohol hydrolases transform HPODE into
trihydroxyoctadecenoates (11).
Lipoxygenases (LOX, linoleate:oxygen oxidoreductase, EC
1.13.11.12) catalyze the enantioselective insertion of molecular
oxygen into a (1Z,4Z)-pentadiene system of polyunsaturated
fatty acids (PUFA) and conjugated cis,trans-diene hydroper-
oxides result.
Multiple isoenzymes of LOX have been described for many
plant species, such as soybean, cucumber, tomato, potato, and
rice (1-4). These LOX enzymes transform linoleic acid into
(S)-9-hydroperoxy-10E,12Z-octadecadienoic acid [(S)-9-HPODE]
and (S)-13-hydroperoxy-9Z,11E-octadecadienoic acid [(S)-13-
HPODE], respectively.
The dioxygenation of linoleic and linolenic acid in plants is
catalyzed by lipoxygenases, and resulting hydroperoxides are
metabolized by further enzymes. Peroxygenases and reductases
catalyze the synthesis of hydroxyoctadecadienoic acids (HODEs)
or hydroxyoctadecatrienoic acids (HOTEs) (5), whereas the
activity of a divinyl ether synthase leads to colnele(n)ic and
etherole(n)ic acids (6-8). The signaling compound jasmonic
acid originates from (S)-13-HPOTE by the activity of an allene
oxide synthase (9). Hydroperoxide lyases catalyze the formation
of aldehydes and ω-oxo acids (10), whereas epoxy alcohol
A lipoxygenase in barley was first reported by Franke and
Freshe in 1953 (12), and subsequently the enzyme was purified
and characterized from ungerminated barley (13-15). However,
these investigations gave attention only to LOX-1, which is
expressed in ungerminated barley. Later, the existence of a
second isoenzyme (LOX-2), which generally appears to develop
only after germination, was documented (16-18). The soybean
and tomato lipoxygenases convert linoleic and linolenic acid
regioselective into (S)-13-hydroperoxides and (S)-9-hydroper-
oxides, whereas the two barley isoenzymes are less regioselec-
tive. LOX-1 catalyzes the dioxygenation of linoleic acid into
(S)-9-HPODE and (S)-13-HPODE in a ratio of ∼80:20, whereas
13-HPODE is the major product formed by LOX-2 (ratio 9-:
13-HPODE ) 30:70) (19-21).
All lipoxygenases characterized in plants catalyze the forma-
tion of (S)-hydroperoxides, including LOX from barley (21, 22).
In contrast, several species of aquatic invertebrates catalyze the
enzymatic oxygenation of their polyunsaturated fatty acids with
(R)-specificity (23-25), and (12R)-LOX has been found in
mammalian tissue (26). Furthermore, soybean lipoxygenase is
able to oxygenate (9Z,12E)-9,12-octadecadienoic acid (cis,trans-
linoleic acid) into (R)-13-HPODE. The rate of the reaction for
the cis,trans-linoleic acid isomer is small (3%) compared to that
for the isomeric cis,cis-substrate but certainly significant (27).
* Author to whom correspondence should be addressed (telephone +49-
30-45080-231; fax +49-30-314-27544; e-mail Leif-A.Garbe@TU-Ber-
lin.de).
10.1021/jf048490s CCC: $30.25
© 2005 American Chemical Society
Published on Web 01/25/2005

HODE in Barley and Malt
J. Agric. Food Chem., Vol. 53, No. 5, 2005 1557
acid methyl esters ( ∼400 µg) were derivatized by treatment with
pyridine (400 µL) and BSTFA (40 µL) at 80 °C for 30 min.
Subsequently, the hydroxy fatty acid methyl esters were analyzed as
trimethylsilyl (Me3Si) ether derivatives.
In addition to free linoleic acid glycerol-esterified PUFAs
are substrates for barley LOXs (21, 22). The facts that
germinating barley LOX-1 and LOX-2 formed both regioiso-
mers of HPODE after incubation with linoleic acid and that
they differ in spatial and temporal expression in barley embryos
(16, 28) suggest that each isoenzyme plays different roles during
germination. 13-LOX is required in the biosynthesis of jas-
monates. The jasmonates are known to influence a wide variety
of physiological processes in plants (29). In oilseed plants 13-
LOX may play a crucial role as the first step of lipid
mobilization to initiate â-oxidation (30, 31).
For GC-MS investigations an HP1 column (cross-linked dimethyl-
polysiloxane, 50 m × 0.2 mm internal diameter, 0.11 µm film, Hewlett-
Packard; temperature program, 1 min at 80 °C, then 10 °C/min to 280
°C) installed in a GC HP 5890 coupled to a mass spectrometer (EI-
MS ionization energy ) 70 eV) was used. The mass spectra were
recorded on an MSD HP 5970. The data were processed with HP
ChemStation [version A.01.03. (1988)]. Carrier gas was He (5.6) at
75 kPa.
An increase of 9-lipoxygenase activity in response to infection
and wounding has been reported for several plant-pathogen
systems (3, 32). However, all physiological functions of the
lipoxygenase pathway are not completely understood.
In contrast to lipoxygenase catalysis, autoxidation forms equal
amounts of racemic 9- and 13-HPODE.
To get more information about the LOX activity in the
different lipid classes during malting, the contents of the regio-
and stereoisomers of the free, triacylglycerol-esterified, and
polar-esterified hydroxyoctadecadienoic acids (HODE) in barley,
germinating barley, and finished malt were characterized.
UV Spectroscopy. The UV spectra were recorded on a Uvikon
spectrophotometer 922 (Kontron Instruments).
High-Performance Liquid Chromatography (HPLC). HPLC was
performed on a Merck HPLC L-7100 with a quaternary solvent gradient
system, which was coupled to a UV detector L-7400. The data were
recorded and analyzed on a Merck Integrator D-7500. The regioisomers
9-HODE-Me and 13-HODE-Me were separated and isolated on a
normal-phase semipreparative column Merck Hibar RT 250-10 Si60
(5 µm); the mobile phase was hexane/diethyl ether (70:30) with a flow
rate of 3 mL/min. The eluate was monitored at 235 nm. Enantiomeric
pairs of the respective HODE-Me were separated with a Chiralcel OD-H
HPLC column (250 × 4.6 mm i.d., 5 µm; J. T. Baker) using hexane/
2-propanol (98:2) as mobile phase at a flow rate of 1 mL/min according
to the method of Martini et al. (33).
MATERIALS AND METHODS
(S)-13-Hydroxy-9Z,11E-octadecadienoic Acid. To a solution of 280
mg (1 mmol) of linoleic acid in 250 mL of 0.1 M borate buffer (pH
9.0) at 4 °C was added soybean lipoxygenase (20 mg, 9.4 units/mg;
Aldrich, Steinheim, Germany) in 5 mL of the buffer. Every 2 min, O2
was bubbled for 20 s through the solution. After 10 min, further soybean
lipoxygenase (20 mg) was added. The mixture was stirred and gassed
with O2 for another 20 min. The suspension was acidified to pH 4
using 2 M H3PO4 and extracted with Et2O (3 × 150 mL), and the
combined organic phases were dried over Na2SO4 and evaporated. The
residue containing the hydroperoxy acid was directly reduced with
NaBH4 (50 mg) in Et2O (30 mL) for 5 h at 4 °C. The crude product
was purified on a silica gel column [20 g of silica gel 60 (0.063-
0.200 mm); solvent 250 mL of PE/EA/acetic acid, 75+25+1].
[13-18O1]-(S)-13-Hydroxy-9Z,11E-octadecadienoic Acid. The syn-
thesis of 13-18O1-labeled (S)-13-HODE was performed utilizing 18O2
(95.4 atom % 18O, Campro Scientific), linoleic acid, and soybean
lipoxygenase. One hundred and fifty milliliters of 0.1 M borate buffer
(pH 9.0) was degassed and N2-flushed in a closed three-neck round-
bottom flask 10 times. Ten milliliters of N2-saturated buffer with 20
mg of soybean lipoxygenase and 200 mg (0.71 mmol) of linoleic acid
dissolved in 10 mL of KOH (1.2%, N2-saturated) were added. Fifty
milliliters of 18O2 gas was added, and the mixture was stirred under
18O2/N2 atmosphere at 4 °C. After 3 h, the incubation was stopped by
acidification with 2 M H3PO4. Extraction, reduction, and purification
were performed according to the synthesis of unlabeled 13-HODE. The
ratio of 18O/16O (S)-13-HODE was 91:9 (GC-MS), and there was 18O-
isotope depletion of the hydroxyl group during the analytical procedure.
This result is in agreement with the literature (34-36).
Chemicals. All chemicals and solvents were purchased from Fluka
(Neu-Ulm, Germany), Sigma-Aldrich (Steinheim, Germany), Merck
VWR International (Darmstadt, Germany), or Roth (Karlsruhe). They
were of analytical grade, HPLC grade, or otherwise purified before
use, if necessary. The soybean lipoxygenase was obtained from Fluka.
18O2 gas was purchased from Campro Scientific (Miamisburg, OH).
Plant Material. Barley (Hordeum Vulgare var. Krona) was germi-
nated in a pilot malthouse. After 4 h of steeping, the degree of steeping
increased to 45% in 48 h. The seeds were germinated at 14 °C and
96% air humidity. After 6 days of germination, samples were kilned
for 18 h at 55 °C following 5 h at 85 °C.
Lipid Extraction and Sample Workup. After milling of the grains
(35-40 g), the nonpolar fraction of barley and malt was isolated by
extraction in a Soxhlet apparatus using n-pentane. After 16 h, the IS
[[13-18O1]-(S)-13-HODE, dissolved in EA] was added to the pentane
solution in the following amounts: for free HODE, 100 µg (barley,
finished malt) and 1 mg (green malt); for triacylglycerol-esterified
HODE, 1,5 mg (barley, finished malt) and 3 mg (green malt). No IS
was added to samples that were subjected to HPLC analysis. The
pentane solution was taken to dryness. To analyze the free HODE, the
residue was esterified by treatment with diazomethane in diethyl ether/
methanol 9:1 (v+v). To quantify the nonpolar-esterified HODE, the
residue was dissolved in 20 mL of MeOH, and 15 mL of 40% KOH/
MeOH was added. The mixture was stirred for 30 min under nitrogen
at 60 °C, followed by the addition of 50 mL of water. The solution
was acidified to pH 3 with 2 M phosphoric acid and extracted with
ethyl acetate (2 × 50 mL). The organic layer was washed with brine
until neutral reaction and dried over Na2SO4. The solvent was
evaporated, and the residue was treated with diazomethane in diethyl
ether/methanol 9:1 (v+v).
(S)-9-Hydroxy-10E,12Z-octadecadienoic Acid. Synthesis of (S)-
9-HODE was performed according to the method of Matthew et al.
(2).
After removal of the nonpolar lipids from milled barley or malt by
Soxhlet extraction, the solid residue was placed in a round-bottom flask
and 1.5 mg of [13-18O1]-(S)-13-HODE was added as IS. The filter cake,
which contains the polar lipids, was treated with KOH/MeOH according
to the pentane fraction under nitrogen. After hydrolysis of the polar
lipid fraction, the fatty acids were esterified by treatment with
diazomethane. The methyl hydroxyoctadecadienoates (HODE-Me) were
purified on a silica gel column [20 g of silica gel 60 (0.063-0.200
mm); solvent, PE/EA, 100 mL of 19+1 (v+v), 100 mL of 18+2, 100
mL of 15+5]. The HODE-Me eluted in the PE/EA 15+5 fraction.
Gas Chromatography (GC)-Mass Spectrometry (MS). HODE-
Me were hydrogenated in H2-satured diethyl ether with 15 mg of
platinum(IV) oxide hydrate as catalyst. The solution was stirred at 0
°C for 90 min, filtered, and taken to dryness. The hydroxyoctadecanoic
(R,S)-13-Hydroxy-9Z,11E-octadecadienoic Acid. Under N2 and at
0 °C 2.61 g of powdered MnO2 (30 mmol) was added to a solution of
(S)-13-HODE (285 mg, 0.96 mmol) in 12 mL of petroleum ether and
stirred for 3 h. The MnO2 was removed by vacuum filtration, and the
solvent was evaporated: 153 mg of 13-keto-9Z,11E-octadecadieonic
acid (KODE) (55%). The reaction was controlled by UV spectroscopy
[λmax 234 nm (HODE) and 280 nm (KODE)].
NaBH4 (83 mg, 2.18 mmol) was dissolved in 5 mL of 0.4 M CeCl3/
MeOH, and 13-KODE (153 mg, 0.52 mmol) was added. The solution
was stirred for 5 min, followed by hydrolysis with 1 N HCl and
extraction with diethyl ether. The organic phases were combined,
washed with NaCl solution until neutral reaction, and dried with Na2-
SO4. The product was controlled by UV spectroscopy and GC-MS.

1558 J. Agric. Food Chem., Vol. 53, No. 5, 2005
Hu¨bke et al.
Figure 1. (a) Low-resolution EI-MS of [13-18O1]-13-HODE isotopic standard (IS). Sample was methylated and silylated before GC-MS analysis. (b)
Low-resolution EI-MS of 13-HODE in malt with [13-18O1]-13-HODE isotopic standard (IS). Samples were methylated, hydrogenated, and silylated before
GC-MS analysis. (c) Low-resolution EI-MS of 9-HODE in malt. Samples were methylated, hydrogenated, and silylated before GC-MS analysis. (d) Total
ion chromatogram (GC-EI/MS) of 9-HODE and 13-HODE in green malt. Samples were methylated, hydrogenated, and silylated before GC-MS analysis
(R,S)-9-Hydroxy-10E,12Z-octadecadienoic Acid. The synthesis of
(R,S)-9-HODE was performed starting with (S)-9-HODE according to
the regioisomer (R,S)-13-HODE.
the purified regioisomers were subjected to a chiral HPLC
analysis (Figures 2). HODE standards were synthesized utilizing
lipoxygenase from tomato [(S)-9-HODE] or soybean [(S)-13-
HODE], and the respective (R)-enantiomers were obtained as
racemates by oxidation (MnO2) and reduction (NaBH4) of the
HODEs.
9-HODE. Investigations of free and esterified HODEs
demonstrated the low amounts of free HODEs in barley and
malt (Table 1). Only 4 mg/kg of 9-HODE (5% of total 9-HODE
lipids) occurs in barley as free acids and increases in green malt
up to 15% (18 mg/kg). During kilning the amount of free
9-HODE decreases, and in the finished malt <2% (1 mg/kg)
was analyzed. In barley and malt ∼50% of the 9-HODE was
characterized in the storage lipids (triacylglycerides) and ∼40%
as polar-esterified 9-HODE.
In barley, enantiomers of 9-HODE occur nearly racemically.
Only a small excess of (R)-9-HODE was detected in all three
lipid classes. During germination the concentration of 9-HODE
increases and is accompanied by a significant alteration of its
enantiomeric ratio in green malt. The enantiomeric excess of
(S)-9-HODE in 6-day-old green malt was determined as
follows: ee (%) ) 78(S) for free 9-HODE, ee (%) ) 14(S) for
triacylglycerol-esterified 9-HODE, and ee (%) ) 16(S) for polar-
esterified 9-HODE. After kilning, the finished malt was analyzed
and a slight excess of (R)-9-HODE [ee (%) ) 6(R)] was found
in the esterified lipids. However, the small amount of free
9-HODE showed an excess of the (S)-configuration [ee (%) )
16(S)] in finished malt (Table 1). The concentrations of the
HODE enantiomers are shown in Figure 3.
13-HODE. According to 9-HODE, the 13-HODE isomer was
analyzed by GC-MS and chiral HPLC (Table 1; Figure 3). In
barley and malt, the concentration of free 13-HODE was low.
This result was comparable to the low content of the free acid
of 9-HODE. The highest concentration of 13-HODE was
detected in the storage lipids. The total concentration of 13-
HODE in all lipid classes increased during germination from
88 mg/kg in barley to 234 mg/kg in the green malt. After kilning,
119 mg/kg was detected in the finished malt. In all lipid classes
of barley, green malt, and finished malt 13-HODE was analyzed
Quantification. All quantitative data presented (mg/kg and ee) are
an average of a duplicate analysis. The standard deviations of the
analytical methods were calculated by analyzing a reference malt 6
times. For GC-MS analysis the standard deviations were as follows:
12% (free), 9% (triacylglycerol), and 11% (polar) for 9-HODE and
7% (free), 5% (triacylglycerol), and 6% (polar) for 13-HODE. Standard
deviations of the chiral HPLC analysis were 2% for free and
triacylglycerol-esterified HODEs and 3% for polar -esterified HODEs.
RESULTS
GC-MS Analysis. An analytical method for the characteriza-
tion of free, triacylglycerol-esterified, and polar-esterified mono-
hydroxyoctadecadienoic acids (HODE) was developed, and data
from barley and malt are presented.
The analysis of HODE was performed by gas chromatogra-
phy-electron impact mass spectrometry (GC-EI/MS) with
isotope dilution assays utilizing [13-18O1]-(S)-13-hydroxy-9Z,-
11E-octadecadienoic acid as standard. The EI-MS fragment ions
(R-cleavage) of 13-HODE (as methylated, hydrogenated, and
silylated derivative) m/z 173 and 174 (analyte, unlabeled) in
comparison to m/z 175 and 176 (standard, labeled) (Figure 1a,b)
were used for quantification. The mass spectrum of 9-HODE
(as methylated, hydrogenated, and silylated derivative) is shown
in Figure 1c. The MS is in agreement with that shown by Yang
et al. (19). The amount of 9-HODE was determined relative to
13-HODE by GC-EI/MS using the total ion current (Figure
1d) and by GC-FID. This method was confirmed by mixing
and analyzing the pure isomers. The classification of HODE
into free, triacylglycerol-esterified, and polar-esterified lipids
was performed as follows: Free HODE were analyzed by direct
methylation of the pentane extract with diazomethane, triacyl-
glycerol HODE after basic hydrolysis of the pentane extract
(in sum with free HODE). The HODE esterified in the polar
lipids were released by treatment of the solid residue of the
Soxhlet extraction with KOH/MeOH and analyzed accordingly.
HPLC Analysis. The regioisomers 9- and 13-HODE were
separated as methyl esters on semipreparative SP-HPLC, and

HODE in Barley and Malt
J. Agric. Food Chem., Vol. 53, No. 5, 2005 1559
Figure 2. (a) HPLC chromatogram of (S)-9-HODE-Me on Chiracel OD-H; detection at 235 nm. (b) Separation of (R,S)-9-HODE-Me on Chiracel OD-H;
detection at 235 nm. (c) Separation of (R,S)-9-HODE-Me from barley on Chiracel OD-H; detection at 235 nm. (d) HPLC chromatogram of (S)-13-
HODE-Me on Chiracel OD-H; detection at 235 nm. (e) Separation of (R,S)-13-HODE-Me on Chiracel OD-H; detection at 235 nm. (f) Separation of
(R,S)-13-HODE-Me from green malt on Chiracel OD-H; detection at 235 nm.
DISCUSSION
with an excess of (S)-configuration. The enantiomeric excess
of the (S)-13-enantiomer was highest in green malt, which is in
accordance with the (S)-9-HODE enantiomer. However, in the
polar lipids the concentration of (R)-13-HODE increased from
14 mg/kg in green malt to 20 mg/kg in finished malt (Figure
3c).
Total HODE. In green malt the total HODE concentration
rises to 350 mg/kg, whereby 192 mg/kg of HODE isomers was
formed through the germination process (Table 2). The ratio
of 9-:13-HODE regioisomers was found to be 26:74, which
reflects the regioselectivity of malt LOX-2. However, (R)-HODE
was analyzed with an increase of 42 mg/kg and a ratio of 17:
83 for the 9- and 13-HODE regioisomers.
9- and 13-HPODE are formed by enzymatic and chemical
peroxidation of linoleic acid or its esters. The resulting hydro-
peroxides are very reactive and are transformed, for example,
to hydroxides by peroxidases, peroxygenases, or other reductions
in plants. Therefore, the amount of HODEs should reflect the
generation of linoleic acid hydroperoxides.
Barley. The monohydroxy acids occur nearly racemically
[(R):(S)) = 50:50] in ungerminated barley. The small excess of
the (R)-9-HODE enantiomer indicates an enzymatic degradation
of (S)-9-H(P)ODE, because the formation of the (S)-enantiomer
is catalyzed by LOX activity and autoxidation only forms
racemic mixtures.

1560 J. Agric. Food Chem., Vol. 53, No. 5, 2005
Hu¨bke et al.
Table 1. Free and Triacylglycerol- and Polar-Esterified HODE in
Barley, Green Malt, and Finished Malt (Dry Matter) and Enantiomeric
Ratios of HODE Isomers
9-HODE
S:R (%)
13-HODE
S:R (%)
mg/kg
mg/kg
barley
free
triacyl
polar
4
36
30
70
48:52
47:53
46:54
47:53
3
60
25
88
53:47
53:47
52:48
53:47
total
6-day green malt
free
triacyl
polar
total
18
53
45
89:11
57:43
58:42
62:38
43
147
44
79:21
64:36
68:32
68:32
116
234
finished malt
free
triacyl
polar
total
1
40
30
71
58:42
47:53
47:53
47:53
2
67
50
53:47
60:40
60:40
60:40
119
The analysis of 13-HODE in ungerminated barley demon-
strated an excess of the (S)-enantiomer in all lipid classes.
Although numerous investigations have not shown the existence
of LOX-2 in sound barley, the formation of 13-hydroperoxides
cannot be the result of an autoxidation exclusively. The barley
LOX-1 enzyme catalyzes the oxygenation of linoleic acid into
9- and 13-hydroperoxides in a distinct ratio of 88:12, which
explains the excess of (S)-13-HODE in barley. However, in
consideration of the high amount of (R)-9-HODE and a minor
excess of the (S)-13-HODE isomer, autoxidation is most likely
involved in the formation of H(P)ODEs in ungerminated barley.
Germination. Upon germination a second lipoxygenase
isoenzyme (LOX-2) is reported to develop (16-18).
The 6-day green malt showed a significant increase of the
amounts of the free (S)-9- and (S)-13-HODE enantiomers (Table
3a). The concentrations of (S)-9-HODE rose from 2 to 16 mg/
kg and those of (S)-13-HODE from <2 to 34 mg/kg (Figure
3a). These increases of 14 mg/kg [(S)-9-HODE] and 32 mg/kg
[(S)-13-HODE] are in accordance with the LOX-2 catalysis,
which forms 9- and 13-hydroperoxides in a ratio of 30:70.
Characterization of triacylglycerol-esterified HODE in 6-day
green malt showed an increases of the (S)-enantiomer of 13
mg/kg for 9-HODE and 62 mg/kg for 13-HODE (Figure 3b;
Table 3b). This ratio of 17:83 reflects the regioselectivity of
LOX-2 catalysis. These data suggest the oxygenation of free
and triacylglycerol-esterified linoleic acid during germination
only by LOX-2 activity. However, the formation of H(P)ODEs
in germinating barley seems to be more complex because the
amounts of (S)- as well as (R)-enantiomers increase significantly.
From barley to 6-day green malt the amounts of (R)-9-HODE
showed an increase of 4 mg/kg and that of (R)-13-HODE of 25
mg/kg, which cannot be caused only by autoxidation. A possible
explanation is the simultaneous degradation of autoxidation-
derived (R)-9-HODE via â-oxidation. The â-oxidation of fatty
acids in plants leads to (S)-3-hydroxyacyl-CoA as intermediate
(37, 38), possessing the same absolute configuration as (R)-9-
HODE and (S)-13-HODE, which therefore fit into this degra-
dative pathway. In addition, a trans double bond in 13-HODE
(C-11) is present, and enoyl-CoA isomerase can act directly
without a need of other isomerases (39). (S)-13-Hydroxy-9Z,-
11E-octadecadienoic acid is described as the main product of
lipoxygenase-catalyzed oxygenation of storage lipids in cucum-
ber and soybean, indicating the lipid mobilization during
germination (1, 30, 31).
Figure 3. Concentrations of (a) free HODE isomers, (b) triacylglycerol-
esterified HODE isomers, and (c) polar-esterified HODE isomers in barley,
green malt, and finished malt (dry matter) in milligrams per kilogram.
Table 2. Total Amount (All Three Lipid Classes in Sum) of HODE
Regioisomers in Green Malt and Increase during Germination
HODE concentration (mg/kg)
ratio (9-:13-)
(%)
Σ
HODE
350
9-HODE
116
13-HODE
234
total (analyzed in 6-day
33:67
green malt)
effect of germination
(S)-HODE
+
192
+
+
+
46
39
7
+
+
+35
146
111
24:76
26:74
17:83
+150
(R)-HODE
+
42
The polar-esterified HODEs in barley and malt were quanti-
fied by an indirect method, analyzing the saponified residue of

HODE in Barley and Malt
J. Agric. Food Chem., Vol. 53, No. 5, 2005 1561
LOX isoenzymes decreased during kilning, and only 1-3% of
the total LOX activity developed remained in the cured malt
(40). The decrease of HODEs could be caused by chemical
degradation due to the higher temperatures in the drying process.
The lowering of the content of (S)-13-HODE might be a result
of a â-oxidative metabolism. In comparison, (S)-9-HODE could
be degraded by other downstream enzymes in the LOX pathway
such as the hydroperoxide lyase (from HPODE) or epoxy
alcohol synthase into reactive (S)-9-hydroxy-12,13-epoxy-10E-
octadecenoic acid. The hydroxy epoxy acids are hydrolyzed,
for example, into trihydroxyoctadecenoic acids (41).
Table 3. Amount of HODE Isomers in Green Malt and Increase of
HODE Enantiomers during Germination
HODE concentration (mg/kg)
ratio (9-:13-)
(%)
Σ
HODE
9-HODE
13-HODE
(a) Free HODE
total (analyzed in 6-day
61
18
43
30:70
green malt)
effect of germination
(S)-HODE
+
+
+
54
46
8
+
+
14
14
0
+
+
+8
40
32
26:74
30:70
0:100
(R)-HODE
(b) TG-Esterified HODE
The amount of (R)-13-HODE in the storage lipids rose from
28 mg/kg in barley to 53 mg/kg in the 6-day green malt and
decreased to 27 mg/kg after kilning, whereas the concentration
of (R)-9-HODE changed only slightly during malting, which is
evidence that not just autoxidation causes the high amounts of
(R)-configured HODEs.
total (analyzed in 6-day
200
53
147
27:73
green malt)
effect of germination
(S)-HODE
+104
+
+
17
13
+87
+62
+25
16:84
17:83
14:86
+75
(R)-HODE
+29
+4
(c) Polar-Esterified HODE
total (analyzed in 6-day
89
45
44
51:49
In contrast to the storage lipids, the amount of (R)-13-HODE
in the polar lipids increased during kilning, whereas the (S)-
13-HODE content was retained (Table 4c). The concentration
of polar- and triacylglycerol-esterified (R)- and (S)-9-HODE
changed similarly. The accumulation of (R)-13-HODE in
finished malt was not expected because all other HODEs
decreased during kilning (Figure 3c). A 9Z,12E-linoleic acid
was reported to end up in the (R)-13-HPODE enantiomer by
LOX catalysis in soybean (27), but this linoleic acid isomer
has not been detected in polar lipids in malt so far.
green malt)
effect of germination
(S)-HODE
+
+
+
34
29
5
+
+
+
15
12
3
+
+
+2
19
17
44:56
41:59
60:40
(R)-HODE
Table 4. Amount of HODE Enantiomers in Green Malt and Finished
Malt and Effect of Kilning
HODE concentration (mg/kg)
change during
kilning (%)
green malt
finished malt
(a) Free HODE
ABBREVIATIONS USED
(S)-9-HODE
(R)-9-HODE
(S)-13-HODE
(R)-13-HODE
16
2
34
9
0,6
0,4
1
1
3
−
−
−
−
−
96
80
97
89
95
BSTFA, N,O-bis(trimethylsilyltrifluoroacetamide); EA, ethyl
acetate; ee, enantiomeric excess; GC-MS, gas chromatography-
mass spectrometry; GC-EI/MS, gas chromatography-electron
impact mass spectrometry; H(P)OTE, hydro(pero)xyoctadec-
atrienoic acid; 9-H(P)ODE, 9-hydro(pero)xy-10E,12Z-octadeca-
dienoic acid; 13-H(P)ODE, 13-hydro(pero)xy-9Z,11E-octadec-
adienoic acid; IS, isotopic standard; Me, methyl ester; (SP)-
HPLC, (straight phase) high-performance liquid chromatography;
13-KODE, 13-keto-9Z,11E-octadecadienoic acid; LOX, lipoxy-
genase; PUFA, polyunsaturated fatty acids; PE, petroleum ether.
Σ
HODE
61
(b) TG-Esterified HODE
(S)-9-HODE
(R)-9-HODE
(S)-13-HODE
(R)-13-HODE
30
23
94
53
200
19
21
40
27
107
−37
−
9
−
−
−
57
49
47
Σ
HODE
(c) Polar-Esterified HODE
(S)-9-HODE
(R)-9-HODE
(S)-13-HODE
(R)-13-HODE
26
19
30
14
89
14
16
30
20
80
−
−
46
16
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±
0
43
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