T. Shimamura et al. / Food Chemistry 129 (2011) 1088–1092
1089
reported (Pischetsrieder, Rinaldi, Gross, & Severin, 1998), and AR
from lactose and butylamine has been shown to be an active com-
ponent inducing nuclear NF-jB translocation in macrophages
at 500 rpm for 15 s, the difference in the absorbance between 492
and 600 nm was read on a microplate reader MPR A4i (Tosoh,
Tokyo, Japan) as the absorbance at 0 min. After 20 min at room
temperature, the absorbance difference was read again. An
increase in the absorbance was recorded as the ability of the
sample to reduce XTT (XTT reducibility).
(Wühr, Deckert, & Pischetsrieder, 2010). Thus, AR, which was
formed during the Maillard reaction of lactose, can clearly affect
the quality and function of foods such as milk and dairy products.
At present the formation of AR in the Maillard reaction mixture
has only been shown in model systems such as lactose and
2.5. DNP derivatisation of AR
a
butylamine or N -acetyllysine (Pischetsrieder, Schoetter et al.,
1998; Shimamura et al., 2000), and the generation of AR in milk
has only been presumed (Pischetsrieder, Schoetter et al., 1998).
Presence of AR in milk has not yet been demonstrated because
AR is very labile and difficult to isolate. In this study, we
demonstrated the presence of AR in milk using a 2,4-dinitrophen-
ylhydrazine (DNP) derivatisation method.
The sample (200
5 mg/ml CuSO4 and 800
l
l) was mixed in a test tube with 200
ll of
ll of 7 mM DNP in 2 M HCl and incubated
for 1 h at room temperature. One millilitre of chloroform was
added to this solution and the mixture was vigorously stirred.
The chloroform layer containing the DNP derivative of oxidised
AR (OAR-DNP) was then collected and subjected to HPLC analysis
and purification.
2. Materials and methods
2.6. HPLC conditions
2.1. Reagents and milk sample
HPLC analysis of OAR-DNP was performed using either of the
two systems: Hitachi L-7100 pump, UV–vis detector L-4000 UV
detector, and D-2500 chromato-integrator (Tokyo, Japan) or Shi-
madzu LC-10A pump, SPD-M10A diode array detector, SLC-10A
system controller, and CLASS VP-5 software for data analysis (Kyo-
to, Japan). The HPLC conditions were as follows: column, Wakosil
5SIL (4.6 mm ꢀ 250 mm, Wako Pure Chemical Industries); wave-
length, 525 nm; mobile phase, hexane/chloroform/methanol
Lactose monohydrate was purchased from Nacalai Tesque, Inc.
(Kyoto, Japan). n-Butylamine and DNP were obtained from Wako
Pure Chemical Industries (Osaka, Japan), and XTT and
min from Sigma–Aldrich Co. (St. Louis, MO). Chloroform-d (CDCl3)
was obtained from Cambridge Isotope Laboratories, Inc. (Andover,
MA). All other reagents were of the highest grade commercially
available. Milli-Q water was used throughout all experiments.
Long-life (LL) milk (Nippon Milk Community Co., Ltd., Saitama, Ja-
pan; 140 °C, 3 s) which had an expiration date after 60 days was
purchased from the local supermarket.
a-lactalbu-
(6.5:2.5:1); flow rate, 0.75 ml/min; injection volume, 15 ll.
2.7. Purification of OAR-DNP
Crude OAR-DNP was dissolved in 10 ml of chloroform, and ap-
plied to a silica gel column (10 mm ꢀ 550 mm) packed with 10 g
of Wakogel C300 gel (Wako Pure Chemical Industries), and eluted
with 300 ml of hexane/chloroform/methanol (6.5:2.5:1). The OAR-
DNP fraction, which could be differentiated by its orange colour,
was collected and subsequently purified by normal-phase HPLC
and reversed-phase HPLC to isolate OAR-DNP. The HPLC conditions
were as follows: normal-phase HPLC; column, Cosmosil 5SL-II
(10 mm ꢀ 250 mm, Nacalai Tesque, Inc.); wavelength, 525 nm;
mobile phase, hexane/chloroform/methanol (6.5:2.5:1); flow rate,
2.2. Heating condition
A model solution consisting of a lactose and amino group
compound was heated as described in our previous studies
(Shimamura et al., 2000, 2004). Lactose monohydrate (262 mM)
and butylamine (1.16 M) were dissolved in 1.28 M phosphate buffer
(pH 7.0), and then the final pH was adjusted to 7.0 with phosphoric
acid. In the case of the milk protein, lactose (4.6%) and a-lactalbu-
min (2.3%) were dissolved in 20 mM phosphate buffer (pH 6.7). A
1.5-ml sample tube was filled with 1.2 ml of the model solution
and heated at 100 or 130 °C for the indicated duration using a
dry heater (Dry Thermo Unit DTU-1C, Taitec Co., Saitama, Japan).
Immediately after heating, the sample was cooled on ice to stop
the reaction. The milk was heated in a test tube in order to scale up.
3.0 ml/min; injection volume, 100 ll: reversed-phase HPLC; col-
umn, Cosmosil 5C18-MS-II (10 mm ꢀ 250 mm); wavelength,
525 nm; mobile phase, 60% methanol; flow rate, 2.0 ml/min; injec-
tion volume, 100 ll.
2.8. Instrument analyses
2.3. Preparation of crude AR
1H NMR and 13C NMR spectra, including two-dimensional cor-
Crude AR was prepared from the heated model solution of lac-
tose and butylamine by the method described in our previous
studies (Shimamura et al., 2000, 2004). Briefly, the heated model
solution was extracted three times with a double volume of ethyl
acetate. The ethyl acetate layer was collected and the solvent in
this fraction was evaporated on a water bath at 70 °C under nitro-
gen atmosphere. The residue (crude AR) was used in subsequent
experiments.
relation spectra, were measured with
a JEOL JNM-AL 400
(400 MHz) spectrophotometer (Tokyo, Japan). TMS was used as
an internal standard. Letters s, d, t, q, and m represent singlet, dou-
blet triplet, quartet, and multiplet, respectively, and coupling con-
stants are given in Hz. The IR spectrum was recorded on IR
Prestige-21 (Shimadzu Co.) by the liquid film method.
OAR-DNP derived from lactose-butylamine model system, ((S)-
5,6-dihydroxy-2,3-bis(2,4-dinitrophenylhydrazono)hexanoic acid).
1H NMR (CDCl3) d: 9.18 (d, J = 2.4, 1H, H-3 in DNP), 8.92 (d, J = 2.4,
1H, H-30 in DNP0), 8.61 (dd, J = 9.8 and 2.4, 1H, H-60 in DNP0), 8.51
(dd, J = 9.8 and 2.4, 1H, H-6 in DNP), 8.04 (d, J = 9.8, 1H, H-50 in
DNP0), 7.91 (d, J = 9.8, 1H, H-5 in DNP), 4.27 (m, 1H, H-5 in OAR),
3.84 (dd, J = 11.2 and 3.6, 1H, H-6 in OAR), 3.68 (dd, J = 11.2 and
6.0, 1H, H-6 in OAR), 2.96 (m, 2H, H-4 in OAR). 13C NMR (CDCl3)
d: 175.9 (s, C-1 in OAR), 147.8 (s, C-10 in DNP0), 144.7 (s, C-1 in
DNP), 142.1 (s, C-40 in DNP0), 141.8 (s, C-4 in DNP), 141.4 (s, C-3
in OAR), 134.9 (s, C-2 in OAR), 133.7 (s, C-20 in DNP0), 133.1
2.4. XTT assay
The XTT assay was performed in a 96-well microtiter plate
according to the method described in our previous studies
(Shimamura et al., 2000, 2004). A 60 ll of 0.5 mM XTT prepared
with 0.2 M potassium phosphate buffer (pH 7.0) saturated with
menadione was added into each well. Afterwards, the sample
(40 ll) was added to the well. After mixing on a microplate shaker