Radical-scavenging activity of butylated hydroxytoluene (BHT) and its metabolites

Article abstract of DOI:10.1016/j.chemphyslip.2004.03.005

To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and inhibition rate constants (k_inh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4- hydroperoxy-4-methyl-2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and k _inh were determined from differential scanning calorimetry (DSC) monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2′-azobis(isobutyronitrile) (AIBN) or benzoyl peroxide (BPO) at 70°C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the order BHT (1-2) > BHT-CHO, BHT-OOH (0.1-0.3) > BHT-Q (～0). The n value for BHT with AIBN was approximately 1.0, suggesting dimerization of BHT. The k_inh values declined in the order BHT-Q ((3.5-4.6)×10⁴ M^-1 s^-1) > BHT-OOH (0.7-1.9×10⁴ M^-1 s^-1) > BHT-CHO ((0.4-1.7)×10⁴ M^-1 s^-1) > BHT ((0.1-0.2)×10⁴ M^-1 s^-1). The k _inh for metabolites was greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.

Full text of DOI:10.1016/j.chemphyslip.2004.03.005

Chemistry and Physics of Lipids 130 (2004) 189–195
Radical-scavenging activity of butylated hydroxytoluene
(BHT) and its metabolites
Seiichiro Fujisawaa,∗, Yoshinori Kadomab, Ichiro Yokoec
a
Department of Oral Diagnosis, Meikai University School of Dentistry, 1-1, Keyakidai, Sakado City, Saitama 350-0283, Japan
Division of Biofunctional Molecules, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,
b
101-0062 Tokyo, Japan
Faculty of Pharmaceutical Science, Josai University, 350-0280 Saitama, Japan
c
Received 14 January 2004; received in revised form 8 March 2004; accepted 9 March 2004
Abstract
To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and in-
hibition rate constants (kinh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-
benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4-hydroperoxy-4-methyl-
2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and kinh were determined from differential scanning calorimetry (DSC)
monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2ꢀ-azobis(isobutyronitrile) (AIBN) or benzoyl
peroxide (BPO) at 70 ◦C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the
order BHT (1–2) > BHT-CHO, BHT-OOH (0.1–0.3) > BHT-Q (∼0). The n value for BHT with AIBN was approximately
1.0, suggesting dimerization of BHT. The kinh values declined in the order BHT-Q ((3.5–4.6) × 104 M−1 s−1) > BHT-OOH
(0.7–1.9 × 104 M−1 s−1) > BHT-CHO ((0.4–1.7) × 104 M−1 s−1) > BHT ((0.1–0.2) × 104 M−1 s−1). The kinh for metabolites was
greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT
or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: BHT; BHT metabolites; Radical scavenging activity; Stoichiometric factors; Inhibition rate constants
1. Introduction
and is not carcinogenic for F344 rats or B6C3F1 mice
(National Toxicology Program, 1979). Studies on the
metabolism of BHT in vivo and in vitro have revealed
that the two main processes in mouse and rat are
hydroxylation of the alkyl substituents and oxidation
of the aromatic ring (Takahashi and Hiraga, 1983;
Thompson et al., 1987). A major metabolite of BHT is
3,5-di-tert-butyl-4-hydroxybenzoic acid (Yamamoto
et al., 1991), which may be derived from the corre-
sponding alcohol and aldehyde (BHT-CHO). BHT-Q
Butylated hydroxytoluene (BHT) is a synthetic phe-
nolic antioxidant widely used as a food additive. BHT
has been shown to inhibit the induction of cancer by
a wide variety of chemical carcinogens (Slaga, 1995)
∗
Corresponding author. Tel.: +81-492855511;
fax: +81-492861712.
E-mail address: fujisawa@dent.meikai.ac.jp (S. Fujisawa).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.03.005

190
S. Fujisawa et al. / Chemistry and Physics of Lipids 130 (2004) 189–195
has also been identified as a metabolite of BHT. Ox-
idation of BHT leads to the formation of a number
of minor metabolites, such as BHT-OOH (Matsuo
et al., 1984). Oxidative DNA damage and apoptosis
have been induced by BHT-OOH or BHT-Q, and
also, BHT-OOH and BHT-Q generate intracellular
peroxides (Oikawa et al., 1998). BHT has well-known
adverse effects in enhancing and preventing cancer
development in a variety of tissues and organs, sug-
gesting that BHT acts as prooxidants and/or antioxi-
dants; however, their kinetics of antioxidant activity
remains to be clarified.
There have been numerous quantitative studies, us-
ing induction period methods, of the reactivity of an-
tioxidants (e.g. alpha-tocopherol and BHT) with per-
oxy radicals and of their inhibition of auto-oxidation
(Burton and Ingold, 1981; Niki et al., 1984; Pryor
et al., 1988; Roginsky et al., 2003; Amorati et al.,
2003). These studies have used in vitro systems, be-
cause more biologically relevant systems are complex
and not stoichiometric, making quantitative study
difficult. The relevance of these in vitro studies has
been questioned because of the differences between
cell-free and biological conditions. We thought that
these differences may be associated with the oxy-
gen levels in the different experimental systems. The
oxygen tension under a 15 Torr oxygen atmosphere is
similar to that in many tissues (Kessler et al., 1984;
Burton and Ingold, 1984), suggesting that oxygen is
scarce in living cells. Previous quantitative in vitro
studies were performed under aerobic conditions, and
the efficiency of antioxidants may differ considerably
between aerobic and anaerobic conditions. We re-
cently reported evaluation of the radical-scavenging
ability of polyphenols and beta-carotene-related com-
pounds by induction methods under nearly anaerobic
conditions, indicating that the antioxidant activity
of these compounds was well predicted by a model
based on kinetic and thermodynamic data (Kadoma
and Fujisawa, 2000; Fujisawa et al., 2002).
cals trapped by 1 mole of phenol) and the inhibition
rate of polymerization (kinh) were determined.
2. Experimental
2.1. Materials
BHT and 2,6-di-tert-butyl-4-methoxyphenol (DTB-
MP) were obtained from Aldrich Chem. Co., Mil-
waukee, WI, USA. 2,6-Di-tert-butyl-p-benzoquinone
(BHT-Q) and 3,5-di-tert-butyl-4-hydroxybenzalde-
hyde (BHA-CHO) were purchased from Tokyo Kasei
Kogyo Co., Tokyo, Japan. 2,2ꢀ-Azobis(isobutyronit-
rile) (AIBN) and benzoyl peroxide (BPO) were ob-
tained from Wako Pure Chemical Industries, Ltd.,
Osaka, Japan. 3,5-Di-tert-butyl-4-hydroperoxy-4-me-
thyl-2,5-cyclohexadiene-1-one (BHT-OOH) was syn-
thesized as follows (Scheme 1); 2,6-di-tert-butyl-4-
methylphenol (BHT) (4.4 g, 0.02 mol) was dissolved
in ethanol (50 ml) and a solution of potassium hy-
droxide (2 g in 5 ml water) was added. The flask was
quickly connected to an oxygen buret and the solution
was vigorously agitated. In approximately 30 min,
450 ml (0.02 mol) of oxygen was taken up. The so-
lution was immediately poured into ice water (ap-
proximately 700 ml) and neutralized with acetic acid.
The precipitate that separated was collected on a
filter, washed with water, and dried (4.5 g). Upon
crystallization from n-hexane, colorless needles were
obtained that melted at 116 ◦C. H NMR (500 MHz,
1
CDCl3, ppm): 1.24 (18H, singlet, 25-t-butyl), 1.37
(3H, singlet, 4-Me), 6.57 (2H, singlet. 3,5-H), 7.71
(1H, singlet, 4-OOH).
MMA (Tokyo Kasei Chemical Co., Tokyo, Japan)
was purified by distillation. AIBN and BPO were re-
crystallized from methanol and chloroform/methanol
(1:1 v/v), respectively.
In the present study, we used the induction pe-
riod method with differential scanning calorimetry
to investigate the radical-scavenging activity of BHT
and its metabolites in the polymerization of methyl
methacrylate (MMA) initiated by thermal decompo-
sition of AIBN or BPO. For BHT and each of its
metabolites BHT-CHO, BHT-Q and BHT-OOH the
stoichiometric factor (n) (i.e. the number of free radi-
Scheme 1.

S. Fujisawa et al. / Chemistry and Physics of Lipids 130 (2004) 189–195
191
Fig. 1. Time–exotherm curves of BHT and its metabolites for AIBN (A) and BPO (B): 1 mol% AIBN (or BPO), 0.1 mol% additives, 9.4 M
MMA, at 70 ◦C.
2.2. Induction period and inhibition rate of
polymerization
and the thermal changes induced by polymerization
were recorded for the appropriate periods. The heat
due to polymerization of MMA was 13.0 kcal/mol
in this experiment. The conversion of all samples,
as calculated from DSC thermograms, was 92–96%.
Polymerization curves were derived from DSC ther-
mograms using the integrated heat evoked by the
polymerization of MMA. Typical time-exotherm and
time-conversion curves for BHT and its metabolites
are shown in Figs. 1 and 2. Polymerization curves
break when an inhibitor is consumed. These breaks
are sharp and provide a reliable measure of the induc-
tion period of the inhibitor. The presence of oxygen
retards polymerization because oxygen reacts with
MMA radicals activated by the initiator and then
subsequently produces a non-radical product. Thus,
The induction period and initial rate of polymer-
ization were determined by the method previously
reported (Kadoma and Fujisawa, 2000; Fujisawa
et al., 2002, 2004). In brief, the experimental resin
consisted of MMA and AIBN or BPO with or with-
out additives. AIBN or BPO were added at 1.0 mol%,
and the additives were used at 0.001, 0.01, 0.02,
0.05 and 0.1 mol%. Approximately 10 ␮l of the ex-
perimental resin (MMA: 9.12–9.96 mg) was loaded
into an aluminum sample container and sealed by
applying pressure. The container was placed in a
differential scanning calorimeter (model DSC 3100;
MAC Science Co., Tokyo, Japan) kept at 70 ◦C,
Fig. 2. Time–conversion curves of BHT and its metabolites for AIBN (A) and BPO (B): 1 mol% AIBN (or BPO), 0.1 mol% additives,
9.4 M MMA, at 70 ◦C.

192
S. Fujisawa et al. / Chemistry and Physics of Lipids 130 (2004) 189–195
polymerization of the control was slightly inhib-
ited, even though the reaction was carried out in a
sealed DSC pan, because the pan contained a small
amount of oxygen since it had been sealed in air.
Tangents were drawn to polymerization curves at
an early stage in the run. The induction period of
test compounds was determined from the length of
time between the zero point on the abscissa and the
point of intersection of tangents drawn to the early
stage of polymerization. The induction period (IP)
was calculated from the difference between the in-
duction period of specimens and that of controls. The
2.5. Measurement of the inhibition rate
constant, kinh
When Ri is constant, i.e. when new chains are
started at a constant rate, a steady-state treatment can
be applied and the initial rate of polymerization of
MMA is given by Eq. (3) (Burton and Ingold, 1981;
Niki et al., 1984; Pryor et al., 1988)
Rp = {kp[MMA]Ri1/2}/(2kt)1/2
(3)
where MMA represents methyl methacrylate and kp
and kt are the rate constants for chain propagation and
termination, respectively.
initial rates of polymerization in the absence (Rp )
c
The kp/(2kt)1/2 rate of polymerization of MMA
(9.4 M) by both AIBN (1 mol%) and BPO (1 mol%)
and presence (Rp ) of BHT and its metabolites were
i
calculated from the slope of the plots of the first
linear line of the conversion rate of MMA polymer-
ization (tangent drawn at the early polymerization
stage).
at 70 ◦C was 9.86 × 10−2 M−1/2 s−1/2 (13). The Rp
i
rates are determined by Eq. (4) (Burton and Ingold,
1981; Niki et al., 1984; Pryor et al., 1988)
Rp = {kp[MMA]Ri}/{nkinh[IH]}
(4)
i
2.3. Rate of initiation
in which Rp is the initial rate of inhibited polymer-
i
The induction period method was used to determine
the inhibition rate (Ri) due to the thermal decomposi-
tion of AIBN or BPO, using Eq. (1).
ization. [MMA], n, [IH] and kp are defined above, and
kinh is the rate constant for scavenging (inhibiting) of
MMA radicals by an antioxidant. When polymeriza-
tion of MMA is suppressed and retarded by an antiox-
idant, the rate can be expressed by Eq. (5).
Ri = n[IH]0/[IP]
(1)
Rp /Rp = (2ktRi)1/2/{nkinh[IH]}
(5)
where [IH]0 is the concentration of the inhibitor at
time zero and [IP] is the induction period. DTBM was
used to determine Ri, since its stoichiometric factor,
n, is known to be 2.00 (Burton and Ingold, 1981). In
the case of [MMA] = 9.4 M and [AIBN or BPO] =
0.1 M at 70 ◦C, the induction period method using
DTBM gave the rate of initiation, Ri, at 70 ◦C. The
Ri values of AIBN and BPO were 5.66 × 10−6 and
2.28 × 10−6 M s−1, respectively.
c
i
The rate kinh is given by Eq. (6).
kinh = {Rp (2ktRi)1/2}/{n[IH]Rp }
(6)
c
i
3. Results and discussion
The conversion curves (Fig. 2) were obtained from
exotherm curves (Fig. 1), showing the radical-scaveng-
ing activity of BHT and its metabolites. The inhibi-
tion polymerization curves show a break when the
antioxidant has been completely consumed (Fig. 2).
These breaks are very sharp and provide a reliable
measure of the stoichiometric factor (n) of antiox-
idants. Plots of induction period or Rp /Rp versus
concentrations of antioxidants are shown in Fig. 3(A)
and (B), respectively. Plots of induction period ver-
sus concentration are straight lines, as predicted by
Eq. (2). The stoichiometric factors (n) of antioxidants
calculated by using Eq. (2) are listed in Table 1.
2.4. Measurement of stoichiometric factors (n)
The relative n value in Eq. (2) can be calculated from
the induction periods in the presence of inhibitors.
n = Ri[IP]/[IH]
(2)
c
i
where [IP] is the induction period in the presence of
an inhibitor. The number of moles of peroxy radicals
trapped by the relevant phenol is calculated with re-
spect to 1 mole of inhibitor moiety unit.

S. Fujisawa et al. / Chemistry and Physics of Lipids 130 (2004) 189–195
193
Fig. 3. Induction period or Rp /Rp as a function of mol% for BHT and its metabolites in the AIBN (A) and BPO (B) MMA system:
c
i
1 mol% AIBN (or BPO), 0.1 mol% additives, 9.4 M MMA, at 70 ◦C.
Inhibition rate constants (kinh) calculated by using
Eq. (6) are also listed in Table 1, where the value of
kt of 3.7 × 107 M−1 s−1 was estimated during MMA
polymerization (Fujisawa et al., 2002). The n of BHT
for BPO (approximately 2.0) was two-fold greater
than that for AIBN (approximately 1.0). Similarly,
the n values of BHT-CHO and BHT-OOH for BPO
(n = 0.2–0.3) were approximately 2–3-fold greater
than those for AIBN (n = 0.1). The n of BHT-Q was
close to zero in both initiator systems. The radical
(cyanoisopropyl radical) derived from AIBN belongs
to the alkyl radicals (R•) under anaerobic conditions,

194
S. Fujisawa et al. / Chemistry and Physics of Lipids 130 (2004) 189–195
Table 1
where Rp represents the initial rate of polymerization
with an antioxidant and Ri represents the rate of initi-
ation.
i
Kinetic parameters characterizing the chain-breaking antioxidant
capability of BHT and its metabolites during polymerization of
methyl methacrylate (MMA) by AIBN or BPO at 70 ◦C
The kcl value for control, BHT and BHT-Q for BPO
was calculated as 594, 509 and 405, respectively. The
kcl for BHT and BHT-Q was reduced by approxi-
mately 15% and by approximately 30%, respectively,
compared with the control. It is of interest why the kcl
for BHT was reduced despite the n value of 2. This
may be associated with the electrical stabilization ac-
tivity of BHT during the induction period, because the
para substituent in the intermediate may stabilize the
quinoid form of radicals. The kcl of BHT for AIBN
was similar to that of the control (kcl = 354). In the
BPO system, BHT effectively suppressed the grow-
ing MMA radicals, whereas it did not in the AIBN
system. This may be associated with the formation
of BHT-OOR (a quinone) by the interaction between
BHT and PhCOO• derived from BPO during the in-
duction period.
The kinh values of BHT have been reported to be ap-
proximately 1.4 × 104 M−1 s−1 at 30 ◦C (Burton and
Ingold, 1981), 2.2 × 104 M−1 s−1 at 70 ◦C (Ohkatsu
et al., 1977) and 1.1 × 104 M−1 s−1 at 37 ◦C (Pryor
et al., 1988). The difference between these kinh values
for BHT is rather small despite the different method-
ologies and radical species used. The similarity of the
inhibition rate constants for BHT, which has two ortho
tert-butyl groups, is probably due to steric hindrance of
the formation of the H-bond by tert-butyl substituents
(Pryor et al., 1988; Roginskii, 1990). However, the kinh
value of BHT in the present study was 10-fold less
than those described above. A major reason for this
difference may be the experimental conditions, which
were either aerobic (previous studies) or nearly anaer-
obic (the present study). The phenoxyl radicals of phe-
nolic antioxidants induced by scavenging alkyl per-
oxy radicals (ROO•) derived from the peroxidation of
lipids containing polyunsaturated fatty acids are prob-
ably more stable under nearly anaerobic conditions.
In contrast, phenoxyl radicals can easily react with
oxygen at high oxygen tensions, and consequently
may be regenerated to the parent phenolic compounds
and/or undergo much more oxidation because oxygen
is a biradical. In living organisms, the partial pressure
of oxygen may be rather low (Kessler et al., 1984;
Burton and Ingold, 1984). Pryor et al. previously re-
ported that menadione, a quinone, or beta-carotene, a
Compound
AIBN
BPO
n
kinh
n
kinh
(×10−4
(×10−4
M
−1 s−1
)
M )
−1 s−1
BHT
BHT-Q
BHT-CHO
BHT-OOH
1.18
0.003
0.08
0.08
0.18
3.49
1.27
1.88
2.20
0.04
0.33
0.21
0.12
4.56
0.42
0.67
Computational errors (three determinations) <5%. 9.4 M MMA,
0.1 M AIBN (or BPO); n, stoichiometric factor; kinh, inhibition rate
constant. The determination of n and kinh is described in the text.
whereas that from BPO (PhCOO•, benzoyloxy rad-
ical) belongs to the oxygen centered radicals. The
inhibition function of antioxidants when reacting with
R• was different from that with PhCOO•, and the re-
activity with PhCOO• was greater than that with R•.
The n value for BHT (n < 2) in AIBN-initiated autox-
idation of styrene is due to dimerization of BHT, i.e.
formation of stilbenequinone (Ohkatsu et al., 1977),
since BHT has two bulky tert-butyl substituents on
both ortho positions and a hindered phenol preferably
induces the coupling reaction of phenoxy radicals.
In the present study, the n for BHT in the AIBN
system was approximately 1, suggesting dimerization
of BHT due to the para–para coupling reaction. The
n =∼ 2 of BHT for BPO suggested the reaction of
two PhCOO• with BHT, which could produce an
analogous quinone product, BHT-OOR, similar to
BHT-OOH. The kinh value for BHT and its metabo-
lites in both the BPO and AIBN systems declined in
the order BHT-Q > BHT-OOH > BHT-CHO > BHT
(Table 1). As the n values decreased, the kinh values
increased. The kinh of BHT ((0.1–0.2)×104 M−1 s−1
)
was approximately 20-fold less than that of BHT-Q
((3–5) × 104 M−1 s−1). For sterically similar pheno-
lic compounds, the kinh of BHT-CHO was 4–10-fold
greater than that of BHT, possibly due to electronic
participation of the para substituents. Compared with
its metabolites, BHT scavenged more radicals and its
rate of radical scavenging was greater. The kinetic
chain length (kcl) of 0.1 mol% BHT and BHT-Q for
BPO was calculated by Eq. (7) (Niki et al., 1984).
kcl = Rp /Ri
(7)
i

S. Fujisawa et al. / Chemistry and Physics of Lipids 130 (2004) 189–195
195
Fujisawa, S., Ishihara, M., Kadoma, Y., 2002. Kinetic evaluation of
the reactivity of flavonoids as radical scavengers. SAR QSAR
Environ. Res. 13, 617–627.
Kadoma, Y., Fujisawa, S., 2000. Kinetic evaluation of reactivity of
bisphenol A derivatives as radical scavengers for methacrylate
polymerization. Biomaterials 21, 2125–2130.
Kessler, M., Hoper, J., Skolasinoka, D.K., Klovekorn, W.P.,
Sebening, F., Volkholz, H.J., Beier, I., Kernbach, C., Retting,
C., Richter, H., 1984. Tissue O2 supply under normal and
pathological conditions. Adv. Exp. Med. Biol. 169, 69–80.
Matsuo, M., Mihara, K., Okuno, M., Ohkawa, H., Miyamoto,
J., 1984. Comparative metabolism of 3,5-di-tert-butyl-4-
hydroxytoluene (BHT) in mice and rat. Food Chem. Toxicol
22, 345–354.
Niki, E., Saito, T., Kawakami, A., Kamiya, Y., 1984. Inhibition
of oxidation of methyl linoleate in solution by vitamin E and
vitamin C. J. Biol. Chem. 259, 4177–4182.
Ohkatsu, Y., Haruna, H., Osa, T., 1977. Kinetic evaluation
of reactivity of phenolic activities as antioxidants for
polypropylene. J. Macromol. Sci.-Chem. AII 1975–1988.
Oikawa, S., Nishino, K., Oikawa, S., Inoue, S., Mizutani, T.,
Kawanishi, S., 1998. Oxidative DNA damage and apoptosis
induced by metabolites of butylated hydroxytoluene. Biochem.
Pharmacol. 56, 361–370.
Pryor, W.A., Stricland, T., Church, D.F., 1988. Comparison of
the efficiencies of several natural and synthetic antioxidants
in aqueous sodium dodecyl sulfate micelle solutions. J. Am.
Chem. Soc. 110, 2224–2229.
Roginskii, V.A., 1990. Kinetics of oxidation of polyunsaturated
fatty acid esters inhibited by substituted phenols. Chemical
Abstracts 113, 130.
polyene, did not behave as antioxidants in their system
under aerobic conditions, since the induction period
was too small to measure, suggesting that the kinetic
study of quinones and polyenes might be difficult.
In contrast, with our method under nearly anaerobic
conditions, the radical-scavenging activity of polyenes
could be kinetically evaluated, since the measurement
using a DSC is high sensitive (Fujisawa et al., 2004),
and also that of quinones such as BHT-OOH and
BHT-Q could be evaluated in the present study. Thus,
to mimic biological conditions, the radical-scavenging
activity of antioxidants in vitro should be evaluated
at low oxygen tensions. To be effective radical scav-
engers, monophenolic antioxidants must be able to
trap two radicals but not necessarily able to trap them
quickly. Since the kinetic chain length in biological
membranes containing polyunsaturated fatty acids is
long, ranging from 70 to 100, and is suppressed by
molecular oxygen by a free radical chain mechanism
(Yamamoto et al., 1985), the present findings sug-
gest that BHT is effective as a chain-breaking an-
tioxidant in living systems. In the future, this study
will be continued to develop synthetic and natural
antioxidants.
Acknowledgements
Roginsky, V., Barsukova, T., Loshadkin, D., Pliss, E., 2003.
Substituted p-hydroquinones as inhibitors of lipid peroxidation.
Chem. Phys. Lipids 125, 49–58.
Slaga, T.J., 1995. The inhibition of skin tumor initiation,
promotion, and progression by antioxidants and related
compounds. Crit. Rev. Food Sci. Nutr. 35, 51–57.
This study was supported, in part, by a Grant-in-Aid
from the Minister of Education, Science, Sports and
Culture of Japan.
Takahashi, O., Hiraga, K., 1983. The role of 2,6-di-tert-butyl-4-
methylene-2,5-cyclohexadienone (BHT quinone methide) in
the metabolism of butylated hydroxytoluene. Food Chem.
Toxicol. 21, 279–283.
References
Amorati, R., Ferroni, F., Pedulli, G.F., Valgimigli, L., 2003.
Modeling the co-antioxidant behavior of monofunctional
phenols. Applications to some relevant compounds. J. Org.
Chem. 68, 9654–9658.
Burton, G.W., Ingold, K.U., 1984. ␣-Carotene: an unusual type of
lipid antioxidant. Science 224, 569–573.
Burton, G.W., Ingold, K.U., 1981. Autoxidation of biological
molecules. 1. The antioxidant activity of vitamin E and related
chain-breaking phenolic antioxidants in vitro. J. Am. Chem.
Soc. 103, 6472–6477.
Fujisawa, S., Ishihara, M., Kadoma, Y., 2004. Kinetics of the
radical scavenging activity of ␤-carotene-related compounds.
SAR QSAR Environ. Res.15, 33–41.
Thompson, J.A., Malkinson, A.M., Wand, M.D., Mastovich, S.L.,
Mead, E.W., Schullek, K.M., Laudenschlager, W.G., 1987.
Oxidative metabolism of butylated hydroxytoluene by hepatic
and pulmonary microsomes from rats and mice. Drug Metab.
Dispos. 15, 833–840.
Yamamoto, K., Tajima, K., Takeyama, M., Mizutani, T., 1991.
Further metabolism of 3,5-di-tert-butyl-4-hydroxybenzoic acid,
a major metabolite of butylated hydroxytoluene in rats. Chem.
Pharm. Bull. Tokyo 39, 512–514.
Yamamoto, Y., Niki, E., Eguchi, J., Kamiya, Y., Shimasaki, H.,
1985. Oxidation of biological membranes and its inhibition.
Free radical chain oxidation of erythrocyte ghost membranes
by oxygen. Biochim. Biophys. Acta 819, 29–36.