rats fed on a diet containing gelatin 12 and casein 8 as the protein source. Ratnayake et al. [10] evalu-
ating the effect of different protein sources on the rat serum lipids and liver polyunsaturated fatty acids,
found that gelatin had a hypocholesterolemic effect and also suppressed the incorporation of arachidonic and
docosahexaenoic acids in the liver phospholipids. In that study, the levels of serum triacylglycerols and high
density lipoproteins HDL were 5 and 30 lower in rats fed on the gelatin diets compared with those fed on
the casein diets.
Apoprotein E apoE is a glycoprotein that mediates a high affinity binding of remnant chylomicrons and
very low density lipoprotein VLDL and a HDL sub- class to two different receptors: LDL receptor B, E
receptor and apoE receptor [11]. ApoE deficient mice apoE
− −
fed on a low fat, low cholesterol diet show serum cholesterol levels six to eight times higher than
the wild type mice due to a massive accumulation of remnant lipoproteins [12]. Consequently, these animals
spontaneously develop atherosclerotic plaques, which were similar to human lesions [13].
To obtain further information on the effects of gelatin on the levels of total cholesterol and lipo-
proteins, wild type and apoE
− −
mice were fed a gelatin rich diet for 8 weeks. The influence of gelatin
ingestion on atherogenesis was studied by analyzing lesional development in the proximal aorta of apoE
− −
mice. The results showed that gelatin reduced the serum levels of total and HDL cholesterol and accelerated
atheroma formation in apoE
− −
mice when compared to mice in the control diet 20 of casein.
2. Materials and methods
2
.
1
. Diets The composition of the diets is shown in Table 1.
Diets were prepared every 2 weeks and stored at − 4°C.
2
.
2
. Animals and experimental design The homozygous apoE deficient apoE
− −
mice in C57BL6 background [12,15] were purchased from
Jackson Laboratory USA. Wild type C57BL6 mice were obtained from the ‘Instituto de Cieˆncias Biolo´gi-
cas’, UFMG, Brazil. Fourteen female wild type and 20 apoE
− −
7-week-old female mice were used in the experiment. Wild type mice were fed the control diet
n = 7 or gelatin gel diet n = 7 for 8 weeks. ApoE
−
− mice were fed a control diet n = 10 or gel diet
n = 10 and were distributed in two identical experi- ments using five animals in each group. The animals
were distributed based on their initial weight 16.5 9 1 g for the wild type groups and 18 9 1 g for apoE
− −
groups and initial serum cholesterol 96 9 15 mgdl for the wild type groups and 470 9 60 mgdl for apoE
− −
groups. The mice were housed in plastic cages and kept in a room with a controlled lightdark cycle 12:12
h. Free access to food and water was provided. Indi- vidual weight and food intake were recorded weekly.
Blood samples from the ocular plexus were collected for cholesterol analysis at the beginning and sixth week of
the diet feeding experiment. At the end of the experi- ment, the animals were fasted for eight hours and killed
under ether anesthesia. Blood was collected from the axillary plexus for lipoprotein fractions and amino acid
determinations. The liver was removed, weighted and frozen at − 20°C. The aorta was removed from the
root at the heart to the iliac bifurcation and fixed for histopathological analysis. The relative liver weight
RLW ratio liver weightbody weight × 100 was de- termined and used as an indirect parameter of nutri-
tional status. The animal feeding and treatment protocol was reviewed and approved by the Animal
Care Committee of the Instituto de Cieˆncias Biolo´gicas, UFMG, Brazil.
2
.
3
. Analytical methods Blood samples were centrifuged at 12 000 × g 3500
rpm for 10 min and the sera was used for determina- tion of lipids, protein and free amino acids.
Total cholesterol [16] and triacylglycerols [17] in serum, liver and feces were determined enzymatically
using commercial kits kits Analisa-Belo Horizonte, Brazil. Sera of apoE
− −
mice were diluted 1:2 in 0.85 NaCl before cholesterol determination to keep the ab-
sorbance in the proper range of the test. Total protein and albumin were measured using commercial kits from
Analisa Belo Horizonte, Brazil.
Three samples from serum from each group pooled serum of animals 1 and 2, pooled serum of animals 3
and 4 and serum of animal 5 were used for free amino acids and lipoprotein determination.
Table 1 Composition gkg of control and gel diet given to wild type or
apoE
−−
mice for 8 weeks Gel
Control Component
Maize starch 533
533 100
Casein 200
100 Gelatin
a
100 Sucrose
100 50
Cellulose 50
70 70
Soybean oil 20
20 Vitamin mixture
b
25 25
Mineral mixture
b
Choline 2
2
a
Sigma, St. Louis, MO.
b
AOAC [14].
Table 2 Weight gain g, relative liver weight RLW liver weightbody
weight×100 and blood parameters of wild type and apoE
−−
mice fed diets containing 20 casein control or 10 casein+10 gelatin
gel as the protein source for 8 weeks Parameter
Wild type
a
ApoE
−− b
Gel Control
Control Gel
4.8 9 0.7
c
Weight gain g 3.1 9 0.7
2.0 9 1.0 2.8 9 0.7
3.6 9 0.4 4.0 9 0.3
4.0 9 0.2 RLW
3.5 9 0.6 5.4 9 0.2
5.7 9 0.2 Total proteins
6.4 9 0.4 6.7 9 0.6
gdl 3.7 9 0.1
Albumin gdl 3.8 9 0.2
4.0 9 0.1 4.3 9 0.2
125 9 8 121 9 7
705 9 60 Total cholesterol
558 9 69 mgdl
99 9 20 62 9 12
85 9 22 Triacylglycerols
89 9 29 mgdl
a
n = 7 in each group.
b
n = 5 in each group. A second experiment using the same number of animals yielded similar results.
c
Average 9 S.D. Significant difference PB0.05 from the corresponding control.
root separated by 200 mm intervals were obtained and stained with hematoxylin-eosin. Histological sections
were examined under a light microscope by one individ- ual who had no access to the codes. The lesion size was
evaluated morphometrically using an image analyzer KS 300 program attached to a microcamera and Zeiss
microscope.
2
.
4
. Statistical analysis Evaluation of differences between diets was analyzed
within the genotype wild type or apoE
− −
mice. Statistical analysis were performed using the unpaired
2-tailed Student’s t-test. A value of P B 0.05 was used to define statistical significance. The results were con-
sidered significant at P B 0.05 using unpaired Student’s t-test.
3. Results
