Biochemical Pharmacology, Vol. 39, No. 10, pp. 1597-1601, 1990.
`Printed in Great Britain.
`
`0006-2952/90 $3.00 + 0.00
`© 1990. Pergamon Press pic
`
`COMPARATIVE EVALUATION OF THE ANTIOXIDANT
`ACTIVITY OF a-TOCOPHEROL, a-TOCOPHEROL
`POLYETHYLENE GLYCOL 1000 SUCCINATE AND
`a-TOCOPHEROL SUCCINATE IN ISOLATED HEP ATOCYTES
`AND LIVER MICROSOMAL SUSPENSIONS
`
`RITA CARINI,* GIUSEPPE Pou,* MARIO U. DIANZANI,* SUSAN P. MADDIX,t TREVOR F.
`SLATERt and KEVIN H. CHEESEMANH
`*Department of Experimental Medicine and Oncology, University of Turin, Corso Raffaello. 30, 10125
`Turin, Italy and tDepartment of Biology and Biochemistry, Brunei University, Uxbridge, Middlesex
`UB8 3PH, U.K.
`
`(Received 27 October 1989; accepted 2 January 1990)
`
`Abstract-The antioxidant activity of a-tocopherol polyethylene glycol 1000 succinate (TPGS) and of
`a-tocopherol succinate (TS) has been examined in isolated hepatocytes and microsomal fractions from
`rat liver. Both TPGS and TS require esterase activity to yield free a-tocopherol and, hence, antioxidant
`activity. TPGS and TS consistently exerted a more effective antioxidant protection than an equivalent
`amount of directly-added free a-tocopheroL The low antioxidant efficiency of directly added free a(cid:173)
`tocopherol in such water-based experimental systems as used here seems to be due to its extreme
`hydrophobicity. TPGS, on the other hand, is an extremely hydrophilic compound that is being examined
`as a useful source of a-tocopherol in certain clinical situations and is here shown to be a convenient and
`effective source for experimental studies into lipid peroxidation and antioxidant mechanisms.
`
`The role of free radical mediated reactions such as
`lipid peroxidation in a wide range of tissue injuries
`is a subject of great current interest. One of the main
`experimental approaches to investigate the role of
`such mechanisms in a given model of tissue injury is
`the use of antioxidant free radical scavenging com(cid:173)
`pounds as putative protective agents. Since a-toc(cid:173)
`opherol is the major lipid-soluble chain-breaking
`antioxidant in biological systems [1, 2] it is the pro(cid:173)
`tective agent of choice in model systems where lipid
`peroxidation of cell membranes is under inves(cid:173)
`tigation. Modulation of the membrane levels of a(cid:173)
`tocopherol is not straightforward, however. Deple(cid:173)
`tion of cell membrane a-tocopherol can be achieved
`by feeding a deficient diet over a period of up to 15
`weeks. Enrichment of liver cell membranes with a(cid:173)
`tocopherol can be achieved by i.p. injection of a
`large dose of the compound before killing the animal
`[2, 3] but this technique is quantitatively unpre(cid:173)
`dictable however, and is altogether rather unwieldy.
`The direct addition of a-tocopherol to isolated cells
`or cell membrane suspensions in vitro would be a far
`more convenient way of investigating the antioxidant
`effects of this compound in many experimental
`model systems. Unfortunately, the extreme hydro(cid:173)
`phobicity of a-tocopherol makes the reproducible
`direct addition of it to water-based systems such as
`suspensions of cells or cell membranes very difficult.
`Using this method, a-tocopherol added to these sys(cid:173)
`tems is generally much less efficient as an antioxidant
`
`:j: Correspondence should be addressed to: Dr K. H.
`Cheeseman, Department of Biology and Biochemistry,
`Brunei University, Uxbridge, Middlesex UB8 3PH, U.K.
`
`than endogenous a-tocopherol. The poor anti(cid:173)
`oxidant effect of a-tocopherol added to aqueous
`suspensions of cells and cell membranes seems to be
`due to an uneven dispersion that prevents its efficient
`dissolution in the cell membranes.
`Esters of a-tocopherol that are more hydrophilic
`are available: a-tocopherol succinate (TS) is often
`used in tissue culture media and for direct addition
`to primary incubations of cells. Also available is a(cid:173)
`tocopheryl polyethylene glycol 1000 succinate
`(TPGS) that has been the subject of little exper(cid:173)
`imental attention but which has recently been shown
`to be very useful as a source of vitamin E in children
`with cholestasis who are unable to absorb normal
`dietary vitamin E because of the absence of bile
`salts [4-6]. TPGS, and possibly TS, may also be
`convenient sources of a-tocopherol in experimental
`studies in vitro. TS is still a very hydrophobic com(cid:173)
`pound but TPGS is extremely water-soluble; both
`compounds require de-esterification to reveal the
`free phenol group that is necessary for antioxidant
`activity. In the present paper we have investigated
`the antioxidant activity of TS and TPGS in direct
`comparison with a-tocopherol in isolated hepa(cid:173)
`tocytes and in suspensions of liver microsomes.
`
`MATERIALS AND METHODS
`
`Male Wistar rats, 200-250 g body wt, were
`obtained from Nossan (Correzzana, Italy). They
`were fed on semi-synthetic diet containing 40 I. U.
`of a-tocopherol and 12,000 I.U. of vitamin A/kg,
`with free access to water. Collagenase type I, a(cid:173)
`tocopheryl
`succinate
`(TS)
`and
`bis-(p(cid:173)
`nitrophenyl)phosphate (BNPP) were obtained from
`
`1597
`
`Par Pharm., Inc.
`Exhibit 1010
`Page 001
`
`

`
`1598
`
`R. CARINI et a/.
`
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`1~1/
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`the Sigma Chemical Co. (St. Louis, MO). Alpha(cid:173)
`tocopheryl polyethylene glycol 1000 succinate
`(TPGS) was a gift of Eastman Kodak Co. (Roch(cid:173)
`ester, NY). Hepatocytes were isolated by the col(cid:173)
`lagenase perfusion technique described in [7, 8] with
`minor modifications in order to preserve the glu(cid:173)
`tathione content of the cells [9). Liver cell sus(cid:173)
`pensions (5 x 106 cells/mL) were incubated at 37° in
`stoppered 50 mL flasks in a final volume of 1-2.5 mL
`in a buffered medium [7) at pH 7.4. The a-tocopherol
`content of hepatocytes was evaluated after 30, 60,
`120 and 240 min incubations in the presence of either
`free a-tocopherol, TPGS or TS (each at 100 ttM).
`Alpha-tocopherol and TS were added to the cell
`suspension dissolved in dimethyl sulphoxide (final
`concentration 14 mM), while TPGS was diluted in
`water. Lipid peroxidation was measured as MDA
`production using the TBA test [8) after 1 hr incu(cid:173)
`bation with ADP/iron (2.5 mM/100 ttM) or cumene
`hydroperoxide (200 ttM), or 2 hr incubation with
`CC14 (172 ttM) in presence of free a-tocopherol,
`TPGS or TS (each at 100 ttM) with or without pre(cid:173)
`incubation. The effect of bis-p-nitro-phenyl-phos(cid:173)
`phate (BNPP), an inhibitor of non-specific esterases
`was studied by adding to the hepatocytes 25 ttM
`of the compound 10 min before preincubation and
`incubation procedures.
`Liver microsomes were prepared as described pre(cid:173)
`viously [10) and washed once in 0.15 M KCl. Micro(cid:173)
`somal ascorbate/iron dependent lipid peroxidation
`was evaluated as MDA production using the TBA
`test [8]: microsomes (1.5 mg protein/mL) were pre(cid:173)
`incubated for 30 min with 100 ttM free a-tocopherol
`or a-tocopherol esters followed by 10 min incubation
`with ascorbate-iron (500 ttM/5 ttM) in a system con(cid:173)
`taining 37 mM KCl and 49 mM Tris buffer at pH 7 .4.
`Hepatocyte a-tocopherol was extracted and
`measured essentially as described in [1]. Aliquots of
`hepatocyte suspension were washed once with the
`incubation medium and then mixed with 2 mL of
`100 mM SDS, 4 mL of absolute ethanol and 1 mL of
`n-heptane. After vortex-mixing and a brief cen(cid:173)
`trifugation, then-heptane layer was taken for HPLC
`analysis [1]. TPGS and TS did not interfere with this
`assay. Microsomal proteins were determined by the
`method of Lowry eta!. [11].
`
`RESULTS AND DISCUSSION
`
`The use of a-tocopherol in water-based exper(cid:173)
`imental models is fraught with problems and it is
`often observed that it has very low antioxidant
`activity when added exogenously in comparison with
`a-tocopherol
`that
`is
`endogenously
`present.
`Examples of this are seen in the papers of Cadenas
`et al. [12] and Cheeseman et al. [2] where the effect of
`a-tocopherol added to liver microsomal suspensions
`stimulated to peroxidise by ascorbate-iron and by
`NADPH/ ADP-iron, respectively, was found to be
`much lower than that of a-tocopherol already incor(cid:173)
`porated into the membrane. Since there is no doubt
`that a-tocopherol is an extremely efficient anti(cid:173)
`oxidant it must be concluded that in such systems a(cid:173)
`tocopherol is not incorporated into the membrane in
`a way that permits interaction with lipid peroxyl
`radicals. This may be a consequence of the extremely
`
`30
`
`180
`
`120
`60
`incubation time (min.)
`Fig. 1. Time course for the uptake of free a-tocopherol (e)
`TPGS (.&)and TS (•) by isolated rat hepatocytes measured
`as free a-tocopherol by HPLC. All additions were 100 11M.
`Endogenous a-tocopherol (0.799 ± 0.076 nmol/106 cells)
`was subtracted from each value. Values are means of 3-5
`experiments ±SE.
`
`hydrophobic a-tocopherol molecules aggregating
`upon addition to water to yield micelles external
`to the membrane bilayer. In this paper we have
`investigated the possibility of using less hydrophobic
`derivatives of a-tocopherol as more convenient and
`efficient sources of a-tocopherol for experimental
`models in vitro such as suspensions of isolated cells
`or sub-cellular membrane fractions.
`Reed et at. [13-15] have been foremost in inves(cid:173)
`tigating the role of a:-tocopherol on protecting hepa(cid:173)
`tocytes from oxidative stress. In those studies Reed
`et al. have mostly used TS as an exogenous a:-toc(cid:173)
`opherol source and demonstrated that it can afford
`protection against chemical toxicity. A key point of
`their studies has been the critical importance of the
`external calcium concentration in modulating the TS
`effect and a recent publication [16] has emphasised
`this; in our study the calcium concentration was
`always 1 mM. In considering the value of the esters
`TPGS and TS as exogenous a-tocopherol sources,
`we have examined (a) their efficiency in supplying
`free a:-tocopherol to target cells and cell membranes
`and (b) their efficiency as antioxidants (or, more
`properly, antioxidant precursors) in several pro-oxi(cid:173)
`dant systems.
`When isolated hepatocytes are incubated with free
`a-tocopherol, TPGS or TS, a
`time-dependent
`increase in the free a:-tocopherol content of the cells
`as measured by HPLC was observed (Fig. 1). These
`data should represent, in the case of direct addition
`of free a-tocopherol addition, the actual uptake of
`a-tocopherol by the cells and, in the case of TPGS
`and TS addition, the release of free a-tocopherol
`into the cells by the action of esterases. The ability
`of the intact TPGS molecule to enter cells has been
`demonstrated by Traber et al. [8] using human fibro(cid:173)
`blasts, erythrocytes and an intestinal cell line. Thus
`TPGS may be taken up by hepatocytes as the intact
`molecule and hydrolysed to free a-tocopherol within
`the cell. The rate of a-tocopherol appearance in the
`cells tended to be slower using TPGS or TS compared
`to direct addition of free a-tocopherol (e.g. at 60 min
`incubation) but after 3 hr of incubation these dif(cid:173)
`ferences were not appreciable. It is also apparent
`
`Par Pharm., Inc.
`Exhibit 1010
`Page 002
`
`

`
`Antioxidant activity of water-soluble a-tocopherol esters
`
`1599
`
`Table 1. MDA production stimulated in isolated hepatocytes by ADP-iron, CC14 or cumene hydro(cid:173)
`peroxide and the antioxidant effect of the addition of free a-tocopherol, TGPS or TS (each 100 ,uM)
`with and without pre-incubation
`
`nmols MDA/106 cells(% inhibition)
`
`Treatment
`
`ADP-iron
`
`CC14
`
`Cumene hydroperoxide
`
`Control
`Free a-tocopherol
`With pre-incubation
`Without pre-incubation
`TPGS
`With pre-incubation
`Without pre-incubation
`TS
`With pre-incubation
`Without pre-incubation
`
`4.31 ± 0.31
`3.45 ± 1.o4 (20% )II
`3.50 ± 0.15 (19%)11
`
`1.68 ± 0.08
`
`2.50 ± 0.23
`
`1.05 ± 0.07 (38% ):j:
`0.74 ± 0.31 (56% ):j:
`
`1.48 ± 0.40(41%)§
`2.03 ± o.3o (19% )II
`
`1.52 ± 0.33 (65% )*
`2.50 ± 0.08 ( 42% )§
`
`0.41 ± 0.07 (76% )*
`0.47 ± 0.04 (72% )*
`
`0.77 ± 0.29 (69%)*
`1.12 ± 0.25 (55% )t
`
`1.98 ± 0.35 (54% ):j:
`2.57 ± 0.12 (40%)§
`
`0.70 ± 0.12 (58%)§
`0.39 ± 0.01 (77% ):j:
`
`0.70 ± 0.18 (72%)*
`1.22 ± 0.24 (51% ):j:
`
`The pre-incubation time was 1 hr, incubation with ADP-iron or cumene hydroperoxide was for a
`further 1 hr and with CC14 for a further 2 hr. Values in parentheses are% inhibition relative to controls.
`Values are means ± SE of 8-12 experiments. Statistical significance of difference relative to control
`(Student's t-test): • P < 0.001; t P < 0.002; :j: P <0.01; § P < 0.05; II not significantly different.
`
`that, in all cases, after 3 hr of incubation the hep(cid:173)
`tocytes were able to accumulate nearly 50% of the
`total available a-tocopherol
`in
`the
`incubation
`medium.
`All three compounds afforded some degree of
`antioxidant effect and with all three pro-oxidant
`systems (Table 1). With the tocopherol esters, pre(cid:173)
`incubation (60 min) allowed a significantly greater
`antioxidant effect than did simultaneous addition,
`except in the case of the CC14 system. This enhance(cid:173)
`ment of effectiveness by pre-incubation was not seen
`with the addition of free a-tocopherol. With or with(cid:173)
`out pre-incubation and with all three antioxidant
`forms the CC14 dependent system was consistently
`the most sensitive to inhibition, possibly because it
`exhibited the lowest pro-oxidant activity.
`TPGS and TS consistently provided better pro(cid:173)
`tection against lipid peroxidation than the equivalent
`concentration of free a-tocopherol despite that (at
`least at the beginning of the incubation) less free (and
`therefore active) a-tocopherol would be available in
`the cells. In hepatocytes, depending on which pro(cid:173)
`oxidant stimulus was used, TPGS was 1.3 to 2.9
`times more effective than free a-tocopherol without
`pre-incubation and 1. 7 to 3.3 times more effective
`with pre-incubation. TS was 1.2 to 2.7 times more
`effective than free a-tocopherol without pre-incu(cid:173)
`bation. In hepatocytes TPGS tended to be more
`effective than TS.
`In liver microsomal suspensions the better anti(cid:173)
`oxidant protection afforded by the tocopherol esters
`relative to directly added free a-tocopherol is even
`more striking (Table 2). With 30 min pre-incubation
`TPGS inhibits ascorbate-iron dependent lipid per(cid:173)
`oxidation by 68%, TS inhibits by 47% and free
`a-tocopherol inhibits by a mere 3%. Microsomes
`clearly possess an esterase active towards the toc(cid:173)
`opherol esters since this esterase activity is essential
`for the antioxidant activity of the tocopherol esters
`to be realised by release of the free a-tocopherol.
`Hence, in liposomes TPGS has no antioxidant
`activity (data not shown). The importance of esterase
`
`Table 2. MDA production by rat liver microsomes incu(cid:173)
`bated for 10 min with ascorbate iron (500 ,uM/5 ,uM) after
`30 min pre-incubation with or without a-tocopherol, TPGS
`or TS, each at 100 ,uM. Values in parenthesis are % inhi-
`bition relative to control
`
`Control
`+ a-tocopherol
`+ TPGS
`+ TS
`
`nmol/min/mg
`protein
`
`1.15 ± 0.27
`1.12 ± 0.31 (3% )*
`0.37 ± 0.11 (68% )t
`0.61 ± 0.26 ( 47% )*
`
`Values are means of three experiments in duplicate ±SE.
`• Not significant compared to the control (Student's t(cid:173)
`test).
`t Significant compared to the control (Student's t-test;
`p < 0.05).
`
`activity is also evident from experiments using the
`esterase inhibitor BNPP in isolated hepatocytes.
`After BNPP treatment the hepatocytes converted
`much less of the tocopherol esters to free a-toc(cid:173)
`opherol and reduced their antioxidant efficacy to
`non-significant levels (Table 3).
`Paradoxically, it is clear that TPGS and TS con(cid:173)
`sistently exerted a more effective antioxidant pro(cid:173)
`tection than an equivalent amount of directly added
`free a-tocopherol. It seems likely that the apparent
`lack of activity of directly-added free a-tocopherol
`may be due in some way to its extreme hydro(cid:173)
`phobicity. We envisage that addition of a-tocopherol
`to a water-based system such as a suspension of
`cells or microsomes leads to aggregation of the a(cid:173)
`tocopherol molecules in a micellar form with certain
`physico-chemical characteristics that prevent incor(cid:173)
`poration of the a-tocopherol in the cell membrane
`in an active configuration. From the data shown in
`Fig. 1 it is evident that directly added free a-toc(cid:173)
`opherol is taken up by the cells and at certain time(cid:173)
`points is actually present at concentrations higher
`
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`
`

`
`1600
`
`R. CARINI et a/.
`
`Table 3. a-Tocopherol levels and ADP-iron-dependent MDA production in isolated rat hepatocytes
`after pre-incubation with TPGS or TS: effect of bis-(p-nitro-phenyl) phosphate (BNPP)
`
`a-Tocopherol
`
`Lipid peroxidation/(% inhibition)
`
`without BNPP
`
`with BNPP
`
`without BNPP
`
`with BNPP
`
`Control
`+TPGS
`+ TS
`
`0.80 ± 0.08
`2.89 :±: 0.34*
`3.20 :±: 0.55t
`
`0.56 :±: 0.27
`0.96 ± 0.28:j:
`0.79 ± 0.29:j:
`
`4.31 :±: 0.31
`1.52 ± 0.33 (65% )*
`1. 98 ± 0.35 (54% )t
`
`4.98 :±: 0.49
`4.07 :±: 0.24 (18% ):j:
`3.88 ± 0.23 (22%):j:
`
`Results are nmoles of a-tocopherol or MDA per 106 cells and (in parentheses) percentage inhibition
`of lipid peroxidation by tocopherol esters; they are the mean values ( ±SE) from three experiments.
`Student's t-test: * P < 0.001; t P < 0.01; + no significant difference compared to the control.
`Hepatocytes were treated for 10 min with 25 ,uM BNPP, pre-incubated for 1 hr with 100 ,uM a(cid:173)
`tocopherol esters and then incubated for 1 hr with ADP-iron (2.5 ,uM/0.1 ,uM) at which time lipid
`peroxidation was measured as MDA production.
`
`than that of a-tocopherol derived from the esters. It
`must be deduced that the directly-added a-toc(cid:173)
`opherol is either not in the right location or not in
`an active configuration in the membrane. In the
`former case one may imagine that free a-tocopherol
`added directly to hepatocytes could accumulate in a
`cell membrane not undergoing lipid peroxidation
`while the opposite may be true for TPGS and TS.
`This could be due to the tocopherol esterase activity
`being located in the same membrane as that under(cid:173)
`going lipid peroxidation (e.g. endoplasmic retic(cid:173)
`ulum). However, this would not explain the result
`seen with the microsomal suspension where a-toc(cid:173)
`opherol is added directly to the target membrane.
`Thus, it appears that directly added free a-toc(cid:173)
`opherol is associating with or dissolving in the mem(cid:173)
`brane in an inactive configuration, possibly still in
`aggregates. Our data are in accord with those of
`Cadenas et al. [12] who also concluded that a-toc(cid:173)
`opherol added exogenously is ineffective because it
`is not properly incorporated into the membrane.
`Moreover, they postulated that a-tocopherol might
`need to be incorporated into specific sites in the
`membrane, i.e. near to the sites of radical produc(cid:173)
`tion. The efficacy of TPGS and TS might be due
`to the gradual release of a-tocopherol by esterase
`activity at the appropriate site and rate to allow
`dissolution in the membrane in an active con(cid:173)
`figuration, allowing rapid diffusion in the plane of
`the membrane and scavenging of peroxyl radicals.
`rather than forming inactive aggregates.
`Our data are consistent with the recent report of
`Fariss et al. [16] who also found that when adding
`either free a-tocopherol or TS to hepatocytes the
`level of cell protection was greater with TS despite
`that a lower level of unesterified a-tocopherol was
`reached in the cells. They postulate that some cyto(cid:173)
`protective effect might be due to the intact TS mol(cid:173)
`ecule or even the release of succinate. This to us
`appears to be unlikely and we believe the explanation
`presented here for our data is equally applicable to
`their observations.
`Further work is required to understand precisely
`why a-tocopherol is ineffective when added directly
`to aqueous suspensions of cells or cell membranes.
`Meanwhile TPGS is not only finding use in the clini(cid:173)
`cal situation but is also clearly a very convenient and
`
`effective source of active a-tocopherol for hepa(cid:173)
`tocytes and liver microsomes in vitro.
`
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`16. Fariss MW, Merson MH and O'Hara TM, a-tocopherol
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`toxicity under physiological calcium conditions. Tox(cid:173)
`icol Lett 41: 61-75, 1989.
`
`Par Pharm., Inc.
`Exhibit 1010
`Page 005