425

Effect of Brominated Vegetable Oils on

Heart Lipid Metabolism 1
YOLANDA B. LOMBARDO*, a, ADRIANA CHICCO a, MARIA Z. BASiLICOa, CLAUDIO
BERNAL a and RAUL GUTMAN a,b, aDepartment of Biochemistry. University of Litoral,
Santa/re, and bDepartment of Experimental Medicine, Hospitale Italiano, Buenos Aires,
Argentina

ABSTRACT

Normal rats fed for 105 days on an experimental diet made up of standard laboratory chow supple-
mented with 0.5% of a mixture of brominated sunflower-olive oil (BVO) developed a significant
increase in the triacylglycerol content of the heart, liver and soleus muscle compared to controls. In
addition, BVO-treated rats had a decrease in plasma levels of triacylglycerol and total and HDL choles-
terol. Plasma fatty acid levels and plasma post-heparin lipolytic activities, such as H-TGL, LPL, T-TGL
and MGH were similar to those of control animals fed the standard chow alone.
Heart PDH a (active portion of pyruvate dehydrogenase) was dramatically decreased in the BVO-fed
rats. A faster rate of spontaneous lipolysis was recorded in the isolated perfused preparation of hearts
from the experimental animals. The addition of 10 -7 M of glucagon to the perfusate, however, revealed
a lipolytic effect comparable to the one observed in the control rats. In summary, our findings of
normal fatty acids and low triacylglycerol plasma levels associated with normal activities of the various
PHLA (post-heparin lipolytic activity) enzymes suggest that accumulation of triacylglycerol in heart
muscle may not be explained essentially in terms of an elevated uptake and/or increased delivery of
plasma fatty acids or plasma triacylglycerol. A decreased in situ catabolism of tissue triacylglycerol
also appears unlikely because the spontaneous as well as the glucagon induced lipolysis in the heart
both were found to be unimpaired.
Our results suggest that the mechanisms involved in the toxicologic effects of a BVO diet on heart
lipid metabolism could be exerted mainly at the level of triacylglycerol biosynthesis rather than a
derangement in some known step of their catabolic pathway. Additional studies are necessary to
clarify this matter.
Lipids 20:425-432, 1985.

INTRODUCTION t h e p r e s e n t time. T h u s , M u n r o et al (3) o b s e r v e d

B r o m i n a t e d vegetable oils ( B V O ) have b e e n
used as a f o o d additive for a n u m b e r o f years
w i t h t h e purpose of adjusting t h e d e n s i t y o f
essential flavoring oils a n d to e n h a n c e cloud sta-
bility in t h e m a n u f a c t u r e of certain citrus-
flavored beverages. Relatively little i n f o r m a t i o n
is available, h o w e v e r , o n t h e t o x i c o l o g i c a l p r o p -
erties o f these c o m p o u n d s or of t h e b r o m i n a t e d
f a t t y acids themselves, In this regard, M u n r o
et al (1,2) r e p o r t e d i m p a i r e d f o o d u t i l i z a t i o n
a n d g r o w t h r e t a r d a t i o n as well as an enlarged
h e a r t , liver and k i d n e y in y o u n g male rats fed
for 105 days w i t h diets c o n t a i n i n g 2.5% b r o m i -
n a t e d c o t t o n s e e d oil. Microscopic e x a m i n a t i o n
o f t h e involved tissues revealed lipid a c c u m u l a -
t i o n in liver a n d f a t t y d e g e n e r a t i o n involving
t h e e n t i r e m y o c a r d i u m . A l t h o u g h less m a r k e d ,
similar changes were o b s e r v e d w h e n t h e a n i m a l s
were fed w i t h as low as 0.5% BVO.
Only a few b i o c h e m i c a l changes associated
w i t h B V O i n g e s t i o n have b e e n d o c u m e n t e d t o
1 An abstract pertaining to this work was presented
in November 1984 at the IV Congress of the Pan Amer-
ican Association of Biochemical Societies (PAABS),
Buenos Aires, Argentina.
*To whom correspondence should be addressed at
Department of Biochemistry, University of Litoral,
Santiago del Estero 2829, (3000) Santa Fe, Argentina
r e d u c e d liver glucose 6 - p h o s p h a t a s e a n d glucose
6 - p h o s p h a t e d e h y d r o g e n a s e activities after feed-
ing rats w i t h 0.5% b r o m i n a t e d c o t t o n s e e d oil
for 120 days. H e a r t h o m o g e n a t e s o b t a i n e d f r o m
rats fed for o n l y 3 days w i t h 4 0 - 2 5 0 m g / 1 0 0 g
b o d y w e i g h t o f t h e a b o v e B V O s h o w e d a signi-
ficant decrease in t h e u t i l i z a t i o n of p a l m i t i c
acid. More r e c e n t l y , J o n e s et al ( 4 , 5 ) r e p o r t e d
t h a t lipid b o u n d - b r o m i d e was d e t e c t a b l e in
several t i s s u e s ' o f a n i m a l s given BVO, i n c l u d i n g
h e a r t , liver, k i d n e y , muscle a n d fat.
Scant i n f o r m a t i o n is available p r e s e n t l y o n
basic d y n a m i c b i o c h e m i c a l p a r a m e t e r s w h i c h
m a y be h e l p f u l in u n d e r s t a n d i n g t h e m e c h a n i s m s
b y w h i c h these c o m p o u n d s e x e r t t h e i r t o x i c o -
logical effects. The p r e s e n t s t u d y , t h e r e f o r e ,
was a i m e d at g a t h e r i n g i n f o r m a t i o n o n lipid
m e t a b o l i s m b o t h in vivo and in vitro of h e a r t
muscle o b t a i n e d f r o m rats fed over a p e r i o d of
105 days w i t h a diet c o n t a i n i n g 0.5% b r o m i n a t e d
sunflower-olive oil m i x t u r e ( B V O D ) . In this
s t u d y , m e a s u r e m e n t s of t h e f o l l o w i n g p a r a m -
eters were c o n d u c t e d : h e a r t c o n t e n t of triacyl-
glycerol, c h o l e s t e r o l ( t o t a l a n d esterified), gly-
cogen a n d t o t a l ( P D H ) a n d active ( P D H a ) f o r m s
of t h e e n z y m e p y r u v a t e d e h y d r o g e n a s e ; liver
c o n t e n t of b o t h t r i a c y l g l y c e r o l a n d g l y c o g e n

LIPIDS, VOL. 20, NO. 7 (1985)

426 Y.B. LOMBARDO, A. CHICCO, M.Z. BASILICO, C. BERNAL AND R. GUTMAN
and serum levels of triacylglycerol, cholesterol
and fatty acids. Since the reported increase of
triacylglycerol in liver, heart and other extra-
hepatic tissues also could be the result of a lack
of modulation of lipoprotein lipase activities,
plasma post-heparin hepatic and extrahepatic
triglyceride lipase as well as monoglyceride
hydrolyase activities also were investigated.
Finally, and in order to observe the dynamic
behavior of lipid metabolism under controlled
conditions, the effect of glucagon (an agent of
k n o w n lipolytic effects on heart triacylglycerol)
(6-8) was tested in the in vitro perfusion prepara-
tion of hearts obtained from BVO-fed animals.
MATERIALS AND METHODS
Diets and Animals
Weanling male Wistar rats weighing 70-90 g
were used in all studies. Animals were housed in
a controlled temperature (23 C ) a n i m a l room,
with fixed, 24-hr artificial light cycles (12 hr
light followed by 12 hr darkness). Initially they
were allowed a diet of a standard rat laboratory
chow and water. One week after arrival, the
animals were divided randomly into two groups.
The experimental group received the standard
rat laboratory chow (Ralston Purina Company,
St. Louis, Missouri, U S A ) s u p p l e m e n t e d with
0.5% brominated vegetable oils. The brominated
oil was a combination of sunflower and olive
oil obtained through a commercial supplier
(Abbott, trademark Densitol "A", specific
weight 1.305-1.315 at 25 C ) a n d was graded as
edible food. Control rats received the above
mentioned standard laboratory show supple-
mented with 0.5% corn oil (STD). Both diets
provided approximately 365 calories/100 gm of
chow. Food was available ad-libitum, and both
groups of rats were maintained on their respec-
tive diets for an experimental period of 105
days. The weight of each animal was recorded
twice a week throughout the test period. In a
separate experiment the caloric intake and
weight gain of six animals in each group were
assessed twice a week. Caloric intake was esti-
mated as follows: food was weighed before and
after remaining in the cage for 24 hr. After
separation of feces, weight correction for con-
tamination of urine was carried out by drying
the remaining food in an oven, parallel to an
appropriate reference sample. Thus, average
caloric intake per day per animal (calories/day)
was calculated by dividing the total caloric
intake per cage by the number of rats housed
in it.
On the day of the experiment, food was re-
moved at 7 a.m. unless otherwise indicated,
and all experiments were carried out between
LIPIDS, VOL. 20, NO. 7 (1985)
10 a.m. and noon.
Perfusion Technique
The hearts of control (n=18) and experi-
mental (n=8) rats were perfused by a modified
Langendorff technique. Details of the method-
ology used have been given elsewhere (8,9).
Following perfusion for a 20-rain stabilization
period, glucagon (1.6 nmol/min) dissolved in
Krebs-Henseleit buffer was infused continuously
during the next 20 min by means of a Sage
syringe pump model 255-L through the injection
port directly above the aortic cannula. The
effects of glucagon on disappearance of heart
triacylglycerol as well as the glycerol output
were determined in the same experiment. At
the end of the perfusion the heart was quickly
frozen with a Wollenberger (10) clamp which
had been kept cooled in liquid N2. The myo-
cardial tissue was then pulverized with a percus-
sion mortar to a fine powder at liquid N~
temperature. The powder thus obtained was
stored in liquid N 2 for up to 1 mo with no
detectable changes in triacylglycerol concentra-
tion. Glycerol was analyzed at l-rain time
intervals on perfusate samples collected from
the 18th to 40th min in all experiments. The
total net glycerol output attributable to the
glucagon effect was calculated by subtracting
the baseline levels obtained when the infusion of
glucagon was replaced by buffer alone from the
total glycerol response observed in the presence
of this hormone over the same time intervals in
parallel perfusions. In all experiments, the tissue
wet wt/dry wt ratios were obtained by drying
the perfused hearts or a portion of the frozen
heart powder at 125 C for at least 6 hr and
reweighing afterwards. The glycerol output and
heart triacylglycerol content thus are expressed
per gm of dry weight, correcting in this manner
for any differences in heart water content.
Assay of Post-Heparin Plasma Triglyceride Lipase
and Monoglyceride Hydrolase (MGH)
After i.p. pentobarbital (60 mg/kg body wt),
sodium heparin (200 U/kg body wt) was injected
into the jugular veins of 10 rats (5 on STD and
5 on BVOD), and blood samples withdrawn 5-7
rain afterward from the cava. Post-heparin
lipolytic activity (PHLA) as total plasma post-
heparin triglyceride lipase activity (T-TGL)was
measured by the method of Krauss et al (12).
Post-heparin triglyceride lipase activity released
from the liver (H-TGL) was determined by inhi-
bition of lipoprotein lipase (LPL) by protamine
sulfate (11). We have reported previously (12,
13) that values of H-TGL obtained by heparin
sepharose affinity chromatography are com-
parable to those obtained by inhibition of extra-
 BROMINATED OILS AND LIPID METABOLISM
hepatic LPL by protamine sulfate, thus vali-
dating the use of the latter as a reliable method
for measuring post-heparin H-TGL activity.
LPL activity was calculated by subtracting the
activity found in the assay containing protamine
sulfate (H-TGL) from the activity found in the
assay containing fresh rat serum as an LPL
activator (T-TGL). T-TGL and H-TGL activities
were expressed as p m o l of glycerol m1-1 hr -1 .
MGH was assayed by a slight modification of
the method of Vogel et al (15). More details on
all the above enzyme methodologies have been
given elsewhere (15,12).
Preparation of Tissue Extracts and Pyruvate
Dehydrogenase (PDH) Determinations
Hearts were removed from 18 anesthetized
rats (12 on STD and 6 on BVOD) with a Wollen-
berger clamp precooled in liquid N2 and the
frozen tissue pulverized in a mortar as described
above. Pyruvate dehydrogenase activity in crude
homogenates was assayed in a coupled reaction
system with the final formation of acetyl-
hydroxamate (16). Homogenates of frozen rat
heart ventricular muscle ( 2 0.t g) were prepared
at 2-4 C in 0.5 ml of ice cold 20 mM potassium
phosphate buffer pH 7.0 containing 40% (v/v)
glycerol with a teflon glass homogenizer for
30 sec. Finally, the heart homogenate was soni-
cated for 4 x 15 sec (with 30 secpause) at a
setting of five with a Sonifier (heat systems,
Ultrasonics, Inc.) fitted with a microtip.
Total pyruvate dehydrogenase activity in
heart homogenates was assayed after the inactive
phosphorylated form present in the homogenate
was converted to the active non-phosphorylated
form by pre-incubating with 10 mM MgC12 for
60 min at 25 C (16). For the determination of
the active portion of PDH (PDH a) tissue homog-
enates were assayed directly. PDH activity can
be measured either by following the formation
of acetylphosphate and its colorimetric deter-
mination as acetyl hydroxamate (16), or by
observing the decrease in the spectrophoto-
metric absorbance of p-nitroaniline and the
formation of p-nitroacetanilide (17). We have
reported previously (18) that values of PDH a as
measured by the formation of either p-nitro-
acetanilide or acetylhydroxamate are very simi-
lar, thus validating the use of the latter as a
reliable method for measuring either the active
portion or the total pyruvate dehydrogenase
activity. A description of the assay and details
of the methodology used have been given
elsewhere (18).
Analytical
Blood samples obtained from the jugular
vein of fed rats (STD n=10; BVOD n=10) anes-
427
thetized with pentobarbital (60 mg/kg body wt)
were placed immediately in chilled tubes, centri-
fuged at 4 C and the plasma or serum either
used immediately or stored for no longer than
three days at -20 C until assayed. Serum triacyl-
glycerol (19), total cholesterol (Tc) (20), high
density lipoprotein cholesterol (H DL c) (20,21)
and fatty acids (F.A.) (22) were determined by
spectrometric methods. Heart, liver, kidney and
epididymal fat pad weights were determined in
each group of animals. Heart and liver glycogen
was assayed enzymatically as described by
Hulling (23) and the results expressed in terms
of glucose equivalent from glycogen. Shell fish
glycogen yielded equivalent amounts of glucose
when analyzed enzymatically (23), Triacylglyc-
erol content of liver, heart and soleus muscle
samples was determined on homogenates of
frozen tissues powdered by the method of
Laurell (19). Tissue DNA content was measured
by a micro adaptation of the procedure de-
scribed by Richard (24). Heart total and esteri-
fied cholesterol content were measured in an
aliquot of homogenate tissue with acetone:abso-
lute alcohol, 1 : 1, by the method of Leffler (20).
Total protein was assayed by the method of
Lowry, Rosebrough, Furr and Randall (25).
Glycerol in the perfusate was determined enzy-
matically using glycerol dehydrogenase by the
fluorometric method of Davidson and Kayala
(26). Differences between values obtained in
rats fed the standard or the standard supplement
with brominated oil diet were tested for signifi-
cance by the Student's t-test (27).
Triolein, bovine fraction V albumin (essen-
tially fatty acid free) and heparin sodium salt
from porcine intestinal mucosa were purchased
from Sigma Chemical Company, St. Louis,
Missouri. Glucagon was a gift from the Novo
Company. Glycerol monooleate 90% was pur-
chased from Calbiochem, San Diego, California.
The enzymes and cofactors necessary for the
analysis of plasma, tissues and perfusate samples
were obtained either from Boehringer Mannheim
Biochemicals, Indianapolis, Indiana, or from
Sigma Chemical Company, St. Louis, Missouri.
All other chemicals were of reagent grade.
R ESU LTS
Body Weight and Food Intake
Figure 1 shows the weight gain and caloric
intake of rats maintained on the STD or BVOD
diets for 105 days. As can be seen, the BVOD
was accepted readily by the animals; no differ-
ences from controls could be observed regarding
caloric intake or weight gain. No overall effect
on growth could be detected.
LIPIDS, VOL. 20, NO. 7 (1985)
428 Y.B. LOMBARDO, A. CHICCO, M.Z. BASI'LICO,C. BERNAL AND R. GUTMAN

400
In = 101
:7
In = 101
tD
1--"
"1-
_o
300

200

//' . . . . cn:~) . ~-~ I va~ue

Calories/day 75.55t2.891
9 . .
80.06*-3.44
. - .
NS

100

:~ i 6 ~} 10 1'2 1Z, WEEKS ON

DIET

FIG. 1. Body weight and caloric intake of male rats fed brominated sunflower-olive oil
at dietary levels of 0% (a) STD or 0.5% (e) BVOD for 105 days. 1X -+ SEM. Figures in
parentheses indicate the number of rats in each group. NS: nonsignificant. Calories/day indi-
cate average daily caloric intake throughout the experimental period, determined as discussed
in Materials and Methods.

Plasma Lipid Profiles pmol/mg DNA compared to 9.6 + 2.8 found in

Table 1 shows plasma levels of fatty acids,
triacylglycerol, T c and HDL c and the ratio
HDLc/T c in STD and BVOD fed groups. As can
be observed, triacylglycerol, T c and HDL c levels
were significantly lower in the BVOD group,
whereas the HDLc/T c ratio remained within the
range recorded in the STD group.
controls (p < 0.01) (n=8). The kidney relative
weight (g/100 g.b.w.) was increased in the
BVOD group (0.76 + 0.06) (n=5) compared to
controls (n=5) (0.64 + 0.01; p < 0.05). Epi-
didymal fat pad weight (STD n=5, BVOD n=5),
however, remained comparable in both groups
of animals.

Pyruvate Dehydrogenase Activity in Heart Muscle

Weight and Biochemical Tissue Composition
of Various Organs
Weight and composition of liver tissue ob-
tained from rats fed the STD and BVOD diets
are shown in Table 2. A dramatic increase in
triacylglycerol and total protein contents
accompanied by a significant increase in liver
weight was found in the animals fed the BVOD.
Hearts of BVOD-fed rats showed a signifi-
cant increase in both triacylglycerol and esteri-
fled cholesterol content. Furthermore, heart
glycogen was f o u n d to be dramatically decreased
in these animals, whereas total protein, DNA
and heart weight remained comparable to what
was found in the control group fed the STD
diet (Table 3).
Triacylglycerol content in the soleus muscle
(n=5) of BVOD fed rats was elevated: 22.1 + 3.8
Total pyruvate dehydrogenase activity (PDH)
was comparable in both groups of animals
(Table 4). The proportion of PDH in the active
(PDHa) form, however, was markedly lower
(p < 0.00 I) in animals fed the BVOD diet.
Plasma Post-Heparin H-TG L, LPL, T-TG L
and MGH Activities
Plasma post-heparin H-TGL, LPL, T-TGL
and MGH activities were similar in both groups
of animals as indicated by the following results:
STD (n=5), H-TGL: X + SEM 5.14 -+ 0.71; LPL:
1.90 + 0.56; T-TGL: 7.04 + 0.54 pmol glycerol
m1-1 h -1 and MGH: 1165 +- 43 /~mol glycerol
1-1 min-1 ; BVOD (n=5): H-TGL: 5.30 + 0.44;
LPL: 2.65 -+ 0.77; T-TGL: 7.96 -+ 0.83, and
MGH: 1493 + 99.

LIP1DS, VOL. 20, NO. 7 (1985)

 BROMINATED OILS AND LIPID METABOLISM 429
TABLE 1
Plasma Lipid Profiles

Diet

STD zx BVOD 9 P value

Fatty acid (#Eq/l) 349 -+ 29 a (6) 363 -+ 119 (6) NSb

Triacylglycerol (m mol/l) 0.55 -+ 0.07 (9) 0.29 -+ 0.05 (10) < 0.001
Cholesterol (m mol/1) 1.77 + 0.12 (6) 1.11 -+ 0.08 (6) < 0.01
HDL cholesterol (m tool/l) 1.16 -+ 0.12 (6) 0.63 -+ 0.09 (6) < 0.05
HDL cholesterol
cholesterol ratio 53 -+ 3 (6) 52 -+ 4 (6) NS

a~ _+SEM. Figures in parentheses indicate the number of rats used for each determination.
bNS: nonsignificant.
HDL cholesterol: high density lipoprotein cholesterol.
zxSTD: rats fed on the standard laboratory chow supplemented with 0.5% corn oil.
9 BVOD: rats fed on the standard laboratory chow supplemented with 0.5% brominated
sunflower-olive oil.

TABLE 2

Liver Weight and Biochemical Composition

Diet
STD z~ BVOD 9 P value

Organ weight/100 g rat 3.47 +- 0.08 a (6) 4.41 -+ 0.07 (6) < 0.001
DNA(mg/gwet organ) 3.84 -+ 0.20 (5) 3.51 -+ 0.22 (6) NSb
DNA (mg/100 g rat) 14.0 -+ 0.6 (5) 17.7 +- 1.8 (6) NS
Total protein (mg/100 g rat) 718.7 -+ 40.6 (6) 1012.1 -+ 32.0 (6) < 0.001
Glycogen (#mol/100 g rat) 903.3 -+ 57.8 (6) 977.3 +- 33.5 (6) NS
Triacylglycerol 34.7 -+ 2.8 (8) 80.6 -+ 5.2 (8) < 0.001
(/~mol/100 g rat)

a~ +_SEM. Figures in parentheses indicate the number of rats used for each determination.
bNS: nonsignificant.
zxSTD: rats fed on the standard laboratory chow supplemented with 0.5% corn oil.
9 BVOD: rats fed on the standard laboratory chow supplemented with 0.5% brominated
sunflower-olive oil.

Effects of Glucagon on the in vitro Perfused DISCUSSION

Heart Preparation (Table 5)
A f t e r a 3-min w a s h - o u t p e r i o d , the triacyl-
glycerol c o n t e n t o f t h e heart was significantly
higher (p < 0.001) in rats fed the BVOD diet.
After 40 rain o f p e r f u s i o n w i t h b u f f e r alone,
triacylglycerol c o n t e n t fell to c o m p a r a b l e levels
in b o t h g r o u p s o f animals. In parallel experi-
m e n t s t h e a d d i t i o n o f glucagon (1.6 n m o l ] m i n )
f r o m t h e 2 0 t h to 4 0 t h m i n u t e significantly
r e d u c e d (p < 0.05) t h e triacylglycerol c o n t e n t
to a c o m p a r a b l e e x t e n t in b o t h groups. The dis-
a p p e a r a n c e o f heart triacylglycerol during t h e
p e r f u s i o n was closely paralled b y the glycerol
o u t p u t in t h e p e r f u s a t e : X + SEM 6.0 + 0.5
/amol/g dry weight and 5 . 6 + 0.9 /~mol/g dry
weight, respectively, in t h e STD-fed rats com-
pared to 5.2 + 1.7 and 3.8 + 0.5 in the g r o u p o f
rats fed t h e BVOD diet.
Our results indicate t h a t n o r m a l rats readily
a c c e p t e d an e x p e r i m e n t a l diet c o n t a i n i n g b y
weight 0.5% o f a m i x t u r e o f b r o m i n a t e d
sunflower-olive oil, as i n d i c a t e d by a caloric
intake and weight gain w h i c h were similar t o
c o n t r o l animals fed the s t a n d a r d c h o w .
Rats o n the e x p e r i m e n t a l diet revealed a sig-
n i f i c a n t increase in t h e t r i a c y l g l y c e r o l c o n t e n t
in t h e h e a r t , liver and soleus muscle, in c o n t r a s t
to significantly decreased plasma levels o f b o t h
triacylglycerol and t o t a l and H D L cholesterol.
Measured plasma p o s t - h e p a r i n lipolytic activities
such as H-TGL, LPL, T-TGL and MGH r e m a i n e d
u n c h a n g e d . The liver also s h o w e d an increase in
t o t a l p r o t e i n c o n t e n t and organ weight. The
heart e x h i b i t e d a significant decrease in glyco-
gen c o n t e n t , w h e r e a s the organ weight r e m a i n e d
normal.

LIPIDS, VOL. 20, NO. 7 (1985)

430 Y.B. LOMBARDO, A. CHICCO, M.Z. BASILICO, C. BERNAL AND R. GUTMAN

TABLE 3

Heart Weight and Biochemical Composition

Diet
STD zx BVOD 9 P value

Organ weight/lO0 g rat 0.28 • 0.06 a (6) 0.31 • 0.01 (6) NSb
D N A ( m g / g w e t organ) 2.20 + 0.15 (6) 2.21 • 0.19 (6) NS
Total p r o t e i n ( m g / 1 0 0 g r a t ) 224.2 -+ 11.2 (6) 211.6 • 11.5 (6) NS
Glycogen (/.~mol/g wet organ) 15.7 • 1.2 (8) 6.7 • 0.7 (8) < 0.001
Triacylglycerol (/zmol/g 3.4 • 0.2 (6) 5.6 • 0.2 (6) < 0.001
wet organ)
Total cholesterol (~mol/g 5.06 • 0.18 (6) 6.92 • 0.31 (6) < 0.001
wet organ)
Esterified cholesterol (btmol]g 1.86 • 0.21 (6) 4.34 • 0.28 (6) < 0.001
wet organ)
Esterified cholesterol ratio 36 + 3 (6) 61 + 2 (6) < 0.001
Total cholesterol - -

a ~ _+SEM. Figures in parentheses indicate the number of rats used for each determination.
bNS: nonsignificant.
z~STD: rats fed on the standard laboratory chow supplemented with 0.5% corn oil.
9 BVOD: rats fed on the standard laboratory chow supplemented with 0.5% brominated
sunflower-olive oil.

TABLE 4

Pyruvate Dehydrogenase Activity in Heart of Rats Fed STD or BVOD Diets

Diet
STD • (12) BVOD 9 (6) P value

Total PDH (U/g wet weight) 5.50 • 0.32 a 5.15 • 0.13 NSb
Total PDH (U/rag DNA) 2.33 + 0.15 2.42 +- 0.19 NS
PDH a (U/g wet weight) 3.84 • 0.23 2.32 • 0.07 < 0.001
PDHa(U/mg DNA) 1.76 • 0.17 1.08 -+ 0.08 < 0.01
PDH a (% of total) 71 • 3 45 • 2 < 0.001

a ~ • SEM. Figures in parentheses indicate the number of rats in each group.

bNS: nonsignificant.
PDH: total pyruvate dehydrogenase activity.
PDHa: active portion of pyruvate dehydrogenase.
~STD: rats fed on the standard laboratory chow supplemented with 0.5% corn oil.
eBVOD: rats fed on the standard laboratory chow supplemented with 0.5% brominated
sunflower-olive oil.

A n i n c r e a s e d t r i a c y l g l y c e r o l c o n t e n t in t h e
heart of rats after ingestion of different types
o f b r o m i n a t e d v e g e t a b l e oils in c o n c e n t r a t i o n s
similar to o u r e x p e r i m e n t a l diet h a d b e e n
d e s c r i b e d ' p r e v i o u s l y b y J o n e s et al (4) a n d
M u n r o et al (3). T h e l a t t e r a u t h o r s also r e p o r t e d
a lack of p a l m i t i c acid o x i d a t i o n in h e a r t
h o m o g e n a t e s , w h i c h was a c c o m p a n i e d b y
n o r m a l u t i l i z a t i o n o f p y r u v i c acid (3). T h e c o n -
version o f p y r u v a t e to a c e t y l C o A h a s b e e n
s h o w n to be r e g u l a t e d b y t h e p y r u v a t e d e h y -
d r o g e n a s e c o m p l e x , w h i c h is itself r e g u l a t e d b y
a c o v a l e n t reversible m o d i f i c a t i o n ( 2 8 , 2 9 ) .
M o r e o v e r , t h e p r o p o r t i o n o f P D H in t h e active
f o r m h a s b e e n s h o w n to be u n d e r m e t a b o l i c
a n d h o r m o n a l c o n t r o l in several t i s s u e s , t h e
h e a r t m u s c l e a m o n g t h e m (29). O u r d a t a s h o w e d
similar values for t o t a l P D H activity in t h e
h e a r t s o f r a t s fed t h e STD or t h e B V O D diets.
P D H in t h e active f o r m ( P D H a ) , h o w e v e r , w a s
d e c r e a s e d d r a m a t i c a l l y in t h e g r o u p o f a n i m a l s
fed w i t h t h e BVOD. It is k n o w n t h a t P D H a
levels d e c r e a s e in s t a r v a t i o n d u e to t h e fact t h a t
an i n c r e a s e d p r o v i s i o n o f f a t t y acids a n d k e t o n e
b o d i e s as a l t e r n a t e fuels s u p p r e s s e s p y r u v a t e
o x i d a t i o n in h e a r t m u s c l e ( 3 0 , 3 1 ) . O u r a n i m a l s ,
h o w e v e r , w e r e n o t starved a n d , in a d d i t i o n ,
s h o w e d n o r m a l p l a s m a levels o f f a t t y acids. T h e
i n t e r c o n v e r s i o n o f P D H (active to inactive
f o r m s ) m a y be i n f l u e n c e d b y a h o s t o f f a c t o r s ,
i n c l u d i n g t h e N A D H / N A D +, a c e t y l Co A/Co ASH
a n d A T P / A D P r a t i o s as well as t h e p y r u v a t e

LIPIDS, VOL. 20, NO. 7 (1985)

 BROMINATED OILS AND LIPID METABOLISM 431

TABLE 5
M e t a b o l i c Effects o f Glucagon in the Perfused Rat Heart Preparation

Heart triacylglycerol
(#mol triacylglycerol/g dry wt) Glycerol
Output b 20-40min
Diet Treatment (n) 3 min 20 min 40 min (vmol/g dry wt)

STD ~ None (9) 19.3 • 1.21 15.0 -+ 0.8 14.2 -+ 0.6"~

Glucagon a (9) -- ~ - 9.0 +- 0.5 ~ 5.6 +- 0.9
x
BVOD e None (4) 36.4 -+ 1.0 -I 17.1 • 0.4 18.9 • 1.9
Glucagon a (4) - -- 13.8 -+ 1.2 3.8 • 0.5

aInfusion of 1.6 nmol/min of glucagon was begun after 20 min of equilibration time and
m a i n t a i n e d for 20 rain up to min 40. Hearts w e r e perfused as described in Materials and
M e t h o d s with a Krebs-Henseleit medium containing 2.5 mM Ca 2 +.
b G l y c e r o l w a s a n a l y z e d at l-rain intervals in perfusate samples collected from the 20th
to 40th minute (for details, see methods). The values given are the mean + SEM. Figures in
p a r e n t h e s e s indicate the number of experiments.
x: p < 0.05, xxx: p < 0.001.
~STD: rats fed on the standard laboratory chow supplemented with 0.5% corn oil.
e B V O D : rats fed on the standard laboratory chow supplemented with 0.5% brominated
sunflower-olive oil.

c o n c e n t r a t i o n ( 2 8 , 2 9 ) . T h e r e d u c t i o n of cardiac B V O D s h o w e d a significant decrease in b o t h

palm• o x i d a t i o n in animals fed a B V O D
could be o v e r c o m e in vitro b y t h e a d d i t i o n of
D-L carnitine, A T P a n d C o A S H t o t h e m e d i u m
(3), t h e latter t w o b e i n g k n o w n t o b e closely
related to t h e r e g u l a t i o n of P D H activity.
Finally, t h e r e is t h e possibility t h a t t h e m y o -
card• of B V O D - f e d rats m a y be h y p o x i c ,
as suggested b y M u n r o et al (2), a n d this c o u l d
in t u r n play an i m p o r t a n t role in PDH activity.
H y p o x i a has b e e n s h o w n to s t i m u l a t e glyco-
genolysis (32), a n d it is w o r t h w h i l e to n o t e t h a t
we have f o u n d a n i m p o r t a n t r e d u c t i o n in car-
diac glycogen c o n t e n t in t h e h e a r t s of B V O D -
fed animals.
L i p o p r o t e i n lipase activities in p o s t - h e p a r i n
p l a s m a ( m a i n l y f r o m fat a n d h e a r t o r i g i n ) a r e
c o r r e l a t e d w i t h t h e r e m o v a l rate of p l a s m a tri-
acylglycerol, a n d t h e activities o f t h e s e e n z y m e s
have b e e n s h o w n to be u n d e r h o r m o n a l a n d
n u t r i t i o n a l c o n t r o l ( 3 3 , 3 4 ) . Our results s h o w e d
n o r m a l levels o f H-TGL, LPL, T - T G L and M G H
activities in a n i m a l s fed t h e B V O D . A l t h o u g h
p l a s m a p o s t - h e p a r i n T - T G L activity r e p r e s e n t s
t h e p o o l of h e p a t i c and e x t r a h e p a t i c (e.g.,
cardiac, skeletal muscle, adipose tissue, etc.)
e n z y m e s , we were u n a b l e to d e t e r m i n e t h e
source of t h e e x t r a h e p a t i c f r a c t i o n u n d e r t h e
present experimental conditions.
T h e c o r r e l a t i o n of b o d y w e i g h t w i t h s e r u m
t r i a c y l g l y c e r o l has b e e n established previously
(35). Moreover, t r i a c y l g l y c e r o l levels appear to
b e m o r e closely r e l a t e d to weight gain t h a n to
a c t u a l weight (36). Our results i n d i c a t e d t h a t ,
a l t h o u g h w e i g h t gain a n d caloric i n t a k e were
similar in b o t h g r o u p s o f rats, a n i m a l s fed t h e
p l a s m a triacylglycerol and c h o l e s t e r o l levels.
This could be e x p l a i n e d b y a t o x i c effect o f t h e
b r o m i n e - b o u n d lipid o n t h e synthesis, s e c r e t i o n
or t r a n s p o r t o f l i p o p r o t e i n f r o m t h e liver (5).
Isolated p e r f u s e d h e a r t s of B V O D - f e d ani-
mals s h o w e d a faster rate of s p o n t a n e o u s (basal)
lipolysis t h a n h e a r t s of rats fed t h e s t a n d a r d
c h o w ( T a b l e 5). The a d d i t i o n of 10-7 M gluca-
g o n to t h e p e r f u s a t e , h o w e v e r , r e s u l t e d in a
c o m p a r a b l e l i p o l y t i c effect. Similar results were
f o u n d b y Stam et al (37) in t r i g l y c e r i d e - e n r i c h e d
h e a r t s o b t a i n e d f r o m rats fed a t r i e r u c a t e - r i c h
diet. T h e a u t h o r s h y p o t h e s i z e d t h a t c o n t r o l of
lipolysis in t h e h e a r t m a y b e t h e result of b o t h
t h e rate of r e m o v a l of t h e p r o d u c e d f a t t y acids
a n d t h e provision o f a l t e r n a t e s u b s t r a t e s for
energy m e t a b o l i s m . T h e m e c h a n i s m s regulating
t h e in vivo lipolysis in a n i m a l s fed t h e B V O D
are u n k n o w n at t h e p r e s e n t t i m e . M u r t h y et al
( 3 8 , 3 9 ) r e c e n t l y have r e p o r t e d an increase in
t h e activity o f g l y c e r o l p h o s p h a t e acyltrans-
ferase in t r i g l y c e r i d e - e n r i c h e d h e a r t s of rats
m a d e d i a b e t i c b y alloxan. This increased syn-
thesis of triglycerides c o u p l e d w i t h a n i n h i b i t i o n
o f lipolysis b y f a t t y acids a n d k e t o n e b o d i e s
m a y a c c o u n t for t h e a c c u m u l a t i o n of triglycer-
ides u n d e r t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s .
We are u n a w a r e o f i n f o r m a t i o n o n t h e status of
t h e g l y c e r o t p h o s p h a t e acyltransferase in rats
fed a B V O D diet.
With regard to synthesis, o u r data enlarge
p r e v i o u s o b s e r v a t i o n s in t h a t 0.5% of BVO
a d d e d t o a s t a n d a r d rat c h o w results in an in-
creased fat c o n t e n t n o t o n l y in t h e h e a r t b u t
also in t h e liver a n d skeletal muscle. Our findings

LIPIDS, VOL. 20, NO. 7 (1985)

432 Y.B. LOMBARDO, A. CHICCO, M.Z. BASfLICO, C. BERNAL AND R. GUTMAN
o f n o r m a l f a t t y acids and low triacylglycerol in
plasma, associated w i t h n o r m a l activities o f t h e
11.
various P H L A e n z y m e s , suggest t h a t the accu-
m u l a t i o n o f triacylglycerol in h e a r t muscle m a y
n o t b e e x p l a i n e d in t e r m s o f an elevated u p t a k e 12.
a n d / o r increased delivery to the tissue o f plasma
f a t t y acids or plasma triacylglycerol. Finally, 13.
a decrease o f t h e in situ catabolism o f tissue
triacylglycerol also appears unlikely, because
t h e s p o n t a n e o u s as well as t h e glucagon i n d u c e d 14.
lipolysis in t h e p e r f u s e d heart b o t h were f o u n d 15.
to be u n i m p a i r e d .
Our results suggest that m e c h a n i s m s involved 16.
in t h e t o x i c o l o g i c effect o f a B V O D diet o n
h e a r t lipid m e t a b o l i s m could be e x e r t e d mainly 17.
at the level o f triacylglycerol b i o s y n t h e s i s r a t h e r
t h a n a d e r a n g e m e n t in s o m e k n o w n step o f 18.
t h e i r catabolic p a t h w a y . F u r t h e r studies are
19.
necessary to clarify this.
20.
21.
ACKNOWLEDGMENTS 22.
23.
A. Ferrigutti and L. Argento provided technical 24.
assistance, and Mrs. E. Ferrigutti typed the manu-
script. This investigation was carried out under the 25.
auspices of the CONICET (Consejo Nacional de Inves-
tigaciones Cient[tificas y Tecnoldgicas) Argentina by 26.
Grant Number 10310a[83, Secyt (Secretarfa de Ciencia
y Tecnologia)and Grant Numbers 10031206-103, 104, 27.
and with financial aid from the A.J. Roemmers Foun-
dation for Biochemistry Research Argentina,
28.
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[Received O c t o b e r 12, 1984]