Table 2 Nutritional evaluation of experimental groups
a
Weight gain gday Experimental group
Energy supply weight gain kJg per rat Food intake gday per rat
Control n = 5 35 9 5
2.5 9 0.4 148 9 21
2.7 9 0.4 125 9 19
b
Cholesterol n = 4 32 9 5
21 9 3
bc
Cholesterol+40 coconut 2.0 9 0.4
216 9 28
bc
n = 5 Cholesterol+40 corn n = 5
1.7 9 0.5
bc
21 9 3
bc
256 9 35
bcd
1.7 9 0.2
bc
20 9 3
bc
243 9 48
bcd
Cholesterol+40 olive n = 5 2.6 9 0.2
e
Cholesterol+10 olive n = 4 137 9 18f
e
26 9 4
bce
2.6 9 0.4
e
31 9 4
bef
141 9 19
e
Cholesterol+5 olive n = 5 PB0.0001
ANOVA PB0.0035
PB0.0001
a
Data are shown as means and their standard deviations. Statistical analysis was done using one-way ANOVA and Tukey–Kramer Multiple Comparisons as post hoc test. Different superscripts
b
versus control;
c
versus cholesterol;
d
versus cholesterol+coconut oil;
e
versus cholesterol+ 40 olive; and
f
versus cholesterol+10 olive are significantly different from each other at PB0.001.
2
.
8
. Statistical analysis Results are shown as means and their standard devi-
ations. Analysis of data was done using Instat 2.0 for Macintosh software GraphPad, San Diego, CA, USA.
Some of the analyzed parameters in this study did not show normal distribution according to Shapiro – Wilk
test, or failed in homology of variance. Therefore, analysis of statistically significant differences was car-
ried out using Mann – Whitney U-test for unpaired data, and one-way ANOVA was performed according
to
Kruskal – Wallis test.
Alternatively, one-way
ANOVA, according to Tukey, was carried out and Tukey – Kramer multiple comparison test as post hoc
analysis was used. Differences were considered non-sig- nificant when P \ 0.05. Association between variables
was assessed by Spearman’s rank-order correlation co- efficient r
s
[31].
3. Results
3
.
1
. Dietary characteristics Table 1 summarizes the composition of different
diets. As shown, distinctive general features were lower carbohydrate and protein, higher percentage of fat and
an increase in cholesterol in the different diets, chow being the control. Diets containing different amounts of
fat 5, 10 and 40 were not isocaloric as were diets providing different types of fat at 40 ww content.
Chow and cholesterol diets presented a monounsatu- rated fatty acid content of 33 and almost equal
amounts of polyunsaturated and saturated fatty acids PS ratio 0.9. Coconut oil diet provided a high con-
tent of saturated short chain fatty acids 88.5 and a low content of monounsaturated fatty acids. Corn oil
diet was particularly enriched in linoleic acid and levels of monounsaturation were similar to the control diet.
Olive oil diets were high in monounsaturated fatty acids, differing in their percentages in inverse propor-
tion to the fat content.
3
.
2
. Nutritional e6aluation Table 2 shows that increasing food fat content to
percentages higher than 5 produced significant de- creases P B 0.001 in the food intake, and these de-
creases were the highest with 40 ww rates without any difference among type of oil. However, weight gain
was decreased only in those groups of experimental animals consuming 40 fat containing diets with again
no differences among oils. As results of these data, the ratio of energy supplied related to weight gain was
significantly P B 0.001 higher in animals consuming diets enriched in 40 fat, independent of its source.
3
.
3
. Plasma analysis Table 3 shows the total plasma cholesterol for each
of the seven groups at the end of the experimental period. Dietary cholesterol was a hypercholesterolemic
agent in these animals since plasma cholesterol signifi- cantly P B 0.05 vs. control increased when rats were
fed diets enriched in this compound. When compared to the cholesterol group, simultaneous administration
of saturated fat coconut diet and cholesterol did not increase plasma cholesterol. Corn oil administration
not only prevented hypercholesterolemia caused by cholesterol administration P B 0.001 vs. cholesterol,
but reduced plasma cholesterol to levels lower than control group P B 0.05 vs. control. Olive oil adminis-
tration prevented the hypercholesterolemic effect of cholesterol P B 0.05 vs. cholesterol maintaining simi-
lar values to those found in animals fed on a high carbohydrate diet control group, irrespective of the
dose employed.
The hypercholesterolemic effect of dietary cholesterol Table 3 is mainly due to an increase in LDL-choles-
terol P B 0.001 vs. control, and the hypocholes-
Table 3 Plasma lipid parameters
a
HDL-cholesterol LDL-cholesterol
Experimental condition Triglycerides
Total cholesterol 44 9 3
Control n = 5 19 9 4
67 9 3 41 9 10
Cholesterol n = 4 85 9 7
b
45 9 4 33 9 3
b
62 9 10
b
50 9 4
b
Cholesterol+40 coconut n = 5 23 9 3
c
75 9 5 69 9 14
b
29 9 4
bcd
15 9 3
cd
48 9 7
bcd
44 9 9
cd
Cholesterol+40 corn n = 5 67 9 12
ce
Cholesterol+40 olive n = 5 38 9 5
d
28 9 6
bde
47 9 19 44 9 3
24 9 3
c
Cholesterol+10 olive n = 4 50 9 7
73 9 4 40 9 7
24 9 4
c
69 9 12 43 9 9
c
Cholesterol+5 olive n = 5 PB0.003
ANOVA PB0.002
PB0.0015 PB0.02
a
Data, expressed as mgdl, are means and their standard deviations. Statistical analysis was done using one way ANOVA according to Tukey and Tukey–Kramer Multiple Comparison Test as post hoc analysis. Different superscripts
b
versus control;
c
versus cholesterol;
d
versus cholesterol+coconut oil; and
e
versus cholesterol+corn oil, are significantly different from each other at PB0.05.
terolemic effect of corn oil is due to significant P B 0.001 vs. control reductions in HDL-cholesterol as well
as LDL-cholesterol. LDL-cholesterol levels were lower in the coconut oil group compared to the cholesterol
group P B 0.05 vs. cholesterol which explains the de- crease in total cholesterol observed for this group. The
hypocholesterolemic effect of olive oil is mainly due to a decrease P B 0.05 vs. cholesterol in LDL-cholesterol
and is more pronounced in 5 and 10 olive oil contain- ing diets. LDL-cholesterol in saturated and monounsat-
urated diets was similar and lower than in the cholesterol group. The 40 coconut oil diet was better
at reducing LDL-cholesterol compared to 40 olive oil P B 0.05, and as effective as other doses of olive oil.
HDL-cholesterol levels of monounsaturated fatty acid diets were found among those of saturated and polyun-
saturated diets, reaching the maximum value in animals consuming 10 olive oil diets. The HDL:LDL ratio
was actually more favorable regarding the chow diet 2.3 than the olive oil diets mean of 1.6. Also, the
ratio of 2.2 for the cholesterol + coconut oil diet was relatively favorable when compared with the olive oil
diets.
Plasma triglycerides Table 3 increased P B 0.05 vs. control when rats were fed on cholesterol. This effect
was not enhanced by simultaneous administration of saturated fat. Rats receiving polyunsaturated corn oil
and 5 monounsaturated diets showed a decrease in plasma triglycerides when compared to the cholesterol
group P B 0.05 vs. cholesterol.
Fig. 1A shows that apoA-I used to generate the polyclonal antibody was homogeneously purified and
panel B shows proof that purified rabbit Ig G anti- apoA-I recognized only this apolipoprotein. Plasma
apoA-I concentration Table 4 appeared significantly P B 0.05 vs. control and vs. cholesterol increased in
saturated and monounsaturated fatty acid provided at 40. A 10 monounsaturated diet induced levels of
apoA-I that were significantly P B 0.05 different from the cholesterol and 40 the olive oil group.
Fig. 1. Quality characteristics of apolipoprotein A-I, antibody, hybri- dation conditions and probes used in the present study. A Denatur-
ing polyacrylamide electrophoresis showing homogeneously purified apolipoprotein A-I stained with Coomassie blue. B. Western blot-
ting showing the specificity of inmunopurified rabbit Ig G anti-rat apoA-I. Molecular markers used in A and B were: bovine albumin,
66 kDa; egg albumin, 45 kDa; glyceraldehyde dehydrogenase, 36 kDa; carbonic anhydrase, 29 kDa; and a-lactoalbumin, 14 kDa.
Northern blot analysis reflecting the specificity of hybridation condi- tions for apoA-I C and GPDH D probes. A total of 5 mg of total
RNA was subjected to electrophoresis in a formaldehyde 1 agarose gel. The RNA was transferred to a nylon membrane and hybridized
to radioactive cDNAs. mRNA sizes are indicated on the autoradio- gram in relation to the ribosomal RNAs.
Table 4 Plasma concentration and hepatic expression of rat apolipoprotein
A-I in the different experimental diets
a
Hepatic expression pg of Plasma
Experimental condition
apoA-I mRNAmg total concentration
RNA mgdl
66 9 34 Control n = 5
21 9 4 Cholesterol
49 9 35 16 9 5
n = 4 Cholesterol+40 101 9 34
bc
16 9 3 coconut n = 5
71 9 11 16 9 3
Cholesterol+40 corn n = 5
102 9 12
bce
Cholesterol+40 28 9 11
cde
olive n = 5 89 9 6
cf
Cholesterol+10 27 9 6
c
olive n = 4 Cholesterol+5
89 9 16 19 9 13
olive n = 5 ANOVA
PB0.05 PB0.04
a
Data are means and their standard deviations. Hepatic expres- sions are normalized to the levels of GPDH expression. Statistical
analysis was done using non-parametric one-way ANOVA according to Kruskal–Wallis test and unpaired Mann–Whitney U-test to test
pair-wise differences. Different superscripts
b
versus control;
c
versus cholesterol;
d
versus cholesterol+coconut oil;
e
versus cholesterol+ corn oil; and
f
versus cholesterol+40 olive are significantly differ- ent from each other at PB0.05.
analyzed against coconut or corn oils because the con- ditions are not comparable due to two different
parameters involved: source of fat and fat intake. The degree of relationship among parameters in the
different dietary groups was established using an associ- ation analysis, and the values of Spearman correlation
coefficient r
s
are shown in Table 5. Although a general positive correlation was found between plasma apoA-I
and HDL-cholesterol, the degree of association ap- peared highly variable depending on the diet. While
highly significant correlations were noted for corn and 10 olive oil groups, weaker associations were ob-
served in the remaining groups.
Table 5 also shows the relationship between plasma apolipoprotein A-I concentration and hepatic apoA-I
mRNA. According to the observed correlations, the increase in plasma apoA-I in olive oil containing diets is
strongly related r
s
= 0.97, P B 0.01 to changes in hep-
atic apoA-I mRNA. However this dramatic effect is only observed when supplied dietary fat is 40 ww.
A significant negative correlation r
s
= − 0.97, P B
0.01 was found in the corn oil group and no significant association was observed in the remaining groups.
4. Discussion
