Investigation article

rev chile Med 2017; 145: 1099-1105

Effect of a high fat diet in the process one Laboratory of Biochemistry, Department

of stone formation
of Basic Sciences, Faculty of Medicine,

Catholic University of the Most Holy

bile cholesterol
Conception. Concepcion, Chile.

two School of Biological Sciences, Department

Reginald del Pozo 1, a, LORENA Mardones 1 B, of Pharmacology, University of conception.

Marcelo Villagran 1 B, Katia Muñoz 1 C, susana Roa 1, d, Concepcion, Chile.

FRANCESCA Rozas 1, d, Valeska Ormazabal 2 b, Mirna Muñoz 1, and

to dr.rer.biol.hum.

b dr. biological Sciences.

c Chemical analyst.

tesista nutrition and dietetics.

Effect of a high-fat diet on cholesterol
d

and Magister in clinical biochemistry and

gallstone formation immunology.

This research was partially supported by

background: It is Known That some nutrients play an Important role in the development of
funds from the research direction of the
cholelithiasis. Cholesterol is Carried by micelles and vesicles in the bile. During the first stage of Catholic University of the Most Holy
gallstone formation, cholesterol crystals derived from thermodynamically unstable vesicles. Aim: To Conception (Project din 11/2014). The authors

determine the effect of a high fat diet on blood lipids and bile composition, and Its implication in the declare no conflict of interest.

formation of gallstones. Material and Methods: Two groups of 15 BALB / c mice each, coming
from the same litter, Were Treated with a Control or with a high-fat diet (64% fat and 0.14%
cholesterol). After two months, the animals Were sacrificed, blood and bile samples Were
Obtained. Serum glucose and lipid profiles the Corresponding Were Measured. In bile samples,
cholesterol and phospholipid levels Were Analyzed, and cholesterol transporters (vesicles and received on 31 March
micelles) Were separated by gel filtration chromatography. results: Treated animals Showed an 2017, accepted on 29 August 2017.

87% increase in the total serum cholesterol (p <0.01) 97% increase in HDL-cholesterol (p <0.05)
and a 140% increase in LDL-cholesterol (p <0.05). No changes in serum triglycerides or glucose
mail to: reginald del Pozo Basic Sciences
Were Observed. In bile, 13% increase in biliary cholesterol (p <0.05) but no change was Observed
Department Faculty of Medicine Catholic
in biliary phospholipids. Also, an Increase in biliary vesicular transporters and an Increase of
University of the Most Holy Conception
cholesterol / phospholipid ratio in vesicular transporters Were Observed. riverside alonso 2850. Concepcion, Chile.

rpozo@ucsc.cl

conclusions: A high fat diet May Contribute to the formation of gallstones in our experimental
model.
(Rev Med Chile 2017; 145: 1099-1105)
Key words: cholelithiasis; Dietary Fats; Lipid Metabolism.

L
Metabolic studies in subjects with lipid profile in normal
multifactorial metabolic, diet being an important and hyperlipidemic range, with and without gallstone, but
factoradevelopment of this pathology.
gallstone is considered In this regard, there
a pathology failing to obtain a consistent pattern 1.2. Initially it was
are precedents to suggest that a diet high in fat is a likely postulated that addition of fat in diets designed to reduce
predisposing factor for the development of gallstones. body weight, could reduce the incidence of cholelithiasis
However, still elucidated the impact exerted on high fat by potentiating the gallbladder contractility 3. However,
diets bile lipids, due to differences in lipid metabolism conflicting results have been reported, indicating that the
inherent in the host, at the time of the dietary intervention. high vesicular contractility by high fat intake is not
It has been realized sufficient explanation for the coleli-

1099
Investigation article
lithiasis generated by rapid weight reduction Four.
It has been established that excessive cholesterol diet, is a
predisposing factor to hyperlipidemia, and can induce a
supersaturation of bile and gallstone formation. However,
the type of fat diet, and certain fatty acids specifically, they
can influence cholelithiasis 5-8. Tests performed in an animal
model of rabbits with hypercholesterolemia, indicate that
the intake of a diet high in monounsaturated fatty acids, fail
to normalize the percentages of bile lipids, reducing the
lithogenic index; Conversely, a diet high in fatty acids ω- 3
polyunsaturated index increased lithogenic 9. In hamsters it
has been reported that the incorporation of saturated fatty
acids to the diet increase lithogenic effects 10.11.
Accordingly, the background literature suggests that the
degree of saturation of dietary fatty acids affects the
metabolism of various biliary lipids, altering its secretion
and biliary content, but still is not certain how and when
these changes occur.
The first step in the process of gallstone formation of
cholesterol, is the presence of a bile supersaturated with
cholesterol, followed by formation of cholesterol crystals,
which grow later are added and finally constitute the
macroscopic calculation. The crystallization process is
generated when the capacity of micellar carriers solubilize
biliary cholesterol, forming vesicular transporters, which
are thermodynamically unstable, resulting in the
production of cholesterol crystals exceeds 12-14.
In order to define the influence of dietary lipids on
predisposition to develop cholelithiasis, we studied the
effect of a high fat diet in the lipid composition of the
vesicle carriers and biliary cholesterol micellar BALB / c
mice.
Material and method
Methodological design
The design used in this study was a prospective
experimental quantitative.
Animals
As experimental model 30 male albino mice of strain
BALB / c are used because of their fac-
1100
fat intake and cholelithiasis - r. del Pozo et al
easy handling, present a very similar to the human
genome, and especially for having gallbladder fifteen. The
animals were purchased from the Institute of Public Health
(ISP), 5 weeks old with an average weight of 20 g. They
were randomized in metabolic cages (5 mice / cage) in two
groups: a control group (n = 15), and another treated with
high fat diet (n = 15). The animals were kept in our animal
facility of Faculty, with temperature control at 20 ° C and
cycles of 12 h light / dark. They received food and water a
composition defined ad libitum, with monitoring body
weights and feed intake, at least 2 times a week. After
eight weeks the mice of both groups were killed, after 12
hour fasting, blood and bile samples obtained. The
animals were maintained in accordance with international
standards issued by the "Guide for the Care and Use of
Laboratory Animals," published by the National Institute of
Health 16.
Diet
a commercial brand pellet was used as staple food
(control group) Champion ®, composed of 20.0% protein,
9.2% fat, 54.6% carbohydrate, and 6.2% fiber (Table 1).
The ingredients of this pellet are corn, wheat flour,
soybean meal, wheat bran, fish meal, meat meal, alfalfa
meal, vitamins and minerals (vitamins A, C,
D, E, K, B one, B 6, B 12 niacin, pantothenic acid, biotin, folic
acid, dicalcium phosphate, calcium carbonate,
manganese, cobalt, copper, zinc, iodine, iron, magnesium,
potassium). A diet high in fat, 100 g of staple food, was
added 91 g of lard (brand Palmin ®) and 117 g of butter
(brand Calo ®) ( Table 1).
Analysis of plasma lipids
Plasma lipids (total cholesterol, HDL-cholesterol,
LDL-cholesterol, triglycerides) were quantified using
enzymatic methods using reagents Labtest 17.
Separation of biliary cholesterol transporters:
vesicles and micelles
Due to the small volume of bile in mice (approx. 100
.mu.l / mouse), we perform determinations in a mixture of
bile from 5 mice treated identically. For this purpose, trans-
rev chile Med 2017; 145: 1099-1105
 Investigation article

fat intake and cholelithiasis - r. del Pozo et al

Table 1. Composition of experimental diets Analysis of bile lipids

The cholesterol in the bile, and the chromatographic
Content Control high fat diet (/ fractions, was quantitated using a chemical method 19 and
diet (g / 100 100g)
an enzymatic method 17, respectively. Phospholipids in bile
g)
and chromatographic fractions were determined by
energy (kcal / 100g) 387 674
analysis of inorganic phosphorus twenty.
protein 20.0 6.8

Carbohydrates available 54.6 18,1

Statistic analysis
Total fat: Saturated fat 9.2 63.7
Data are presented as mean values ​± SD. the Student
monounsaturated fat 1.0 34.1
polyunsaturated fat 2.9 21.5 t-test for independent variables with normal distribution
5.3 6.6 was used to establish the levels of statistical significance
cholesterol 0 0,125
between the groups analyzed. P values ​less than 0.05
were considered statistically significant (GraphPad
sodium 0 0,005
software 2017).
Fiber 6.2 2.0

vesicular carriers were separated from the micellar carriers
by gel filtration chromatography 18. Native bile were
centrifuged at 11,200 gx 10 min, and the supernatant 20 ul
were applied on a column (30 x 1.5 cm) containing Bio-Gel
A-5m (rank operation: 10 to 5000 kDa). The
chromatographic fractions (0.3 ml / fraction; flow rate: 0.5
ml / min) were obtained after elution with buffer 20 mM
Tris-HCl (pH 8.0) 3 mM, NaCl 140 mM, sodium azide,
containing 5 mM sodium cholate to prevent disruption of
micellar carriers.
reagents
Were purchased from Sigma (St. Louis, MO) the following
reagents: sodium cholate, cholesterol, phosphatidylcholine, blue
dextran. Bio-Gel A-5m was purchased from Bio-Rad.
results
After 8 weeks of consuming a high fat diet, the mice
showed a significant decrease in body weight of 15%
compared to controls (Figure 1) without

Figure 1. Weights control mice (n = 15) and

treated mice (n = 15) with high-fat diet during the
experimental period. (Average ± SD) * p <0.001;

* * P <0.0001.

rev chile Med 2017; 145: 1099-1105 1101

 Investigation article

fat intake and cholelithiasis - r. del Pozo et al

clear shift in daily food intake (control: 3.3 ± 1.8 g vs.
Treated: 4.3 ± 2.4g).
Plasma lipids showed different behavior after 2 months
of ingestion of a high fat diet (Figure 2). a significant
increase of 87% in plasma total cholesterol levels in the
mice consuming high fat diet, because of an increase of
140% in LDL-cholesterol, and increased 97% in the levels
observed HDL-cholesterol. However, in treated animals,
no significant differences were visualized on triglyceride
concentrations. Also significant differences were observed
tions in blood glucose levels (controls: 87 ± 34 mg / dL vs
treated: 79 ± 36 mg / dL).
Bile lipid treated group showed a significant 13%
increase in cholesterol levels, without alteration in
phospholipid concentrations (Table 2). Moreover, the
treated group had a significant increase in the
concentrations of cholesterol and phospholipids in
vesicular carriers, without significant variations in the
concentrations of phospholipid in micellar carriers (Table
2). Furthermore, the cholesterol ratio doubled /
phospholipids vesicular transporters in animals treated
with a high fat diet (Table 3).

Figure 2. Lipid concentrations in plasma of control

mice treated with high-fat diet for 60 days (mean ±
SD). * P <0.0001;

* * p <0.05; *** p <0.05. Abbreviations: al. total:

total cholesterol. LDL-cholesterol al .: ldl. Hdl-al .:
HDL cholesterol. tag: triglycerides.

Table 2. Concentrations of bile lipids and lipid vesicle carriers (TV) and micellar carriers (TM) present in bile of control mice and mice
treated
with high (mean ± SD) fat diet for 60 days

Mice Cholesterol phospholipids Cholesterol phospholipids Cholesterol phospholipids

Biliary bile (mM) in (mM) in (mM) in TM in TM
(mM) TV TV (mM) (mM)

controls 4.0 ± 0.5 39 ± 12.0 0.4 4.2 ± 2.0 3.7 29.1 ± 2.0

treated with high-fat

4.5 ± 0.4 * 44 ± 15 1.4 7.2 ± 2.8 ** 2.2 28.2 ± 2.8
diet

* p <0.05; ** p <0.005.

1102 rev chile Med 2017; 145: 1099-1105


cholelithiasis - r. del Pozo et al
Table 3. Percentage of cholesterol and / phospholipids ratio (Col / Fos) in vesicular and micellar carriers present in
bile of control mice and
treated with a high-fat diet for 60 days
Mice Vesicular transporters *
% Cholesterol
control 10.5
treated with high-fat diet 38.8
* values ​correspond to bile collection of 5 mice. fat intake and
Discussion
Our results indicate that a high fat diet can play an
important role in the early stages of cholelithiasis.
In this study, one group of mice was subjected to the
intake of a high fat diet (64%, containing 54% saturated
fat, 34% monounsaturated and 10% polyunsaturated fats),
and low cholesterol (0, 14%; 0.18 mg / kcal), for a period
of 2 months. During this period, the treated animals
showed a significant reduction in body weight compared
with the control group, without finding significant
differences in dietary intakes between two groups.
Although epidemiological studies have reported a positive
association between fat intake and obesity twenty-one, Recent
prospective, randomized clinical trials involving individuals,
suggest otherwise 22. Recent studies have shown a greater
body weight loss in subjects consuming high fat diets
(containing 40% to 68% fat), compared with an intake of
low-fat diets (containing ≤
30% fat) for a period of 6 months 23-28.
Recently, a review of several articles comparing body
weight in individuals who consumed high and low-fat
long-term (1 to 6 years) diets showed no significant
differences, but to include only low-calorie diets greater
decrease was observed in body weight of the groups with
high-fat diets 29.
The effect of a high fat diet on lipoprotein levels vary
considerably according to the type of fat consumed. In
general, it has been reported that saturated fatty acids
increase both the total plasma cholesterol and LDL
cholesterol 29.30. In contrast, polyunsaturated fatty acids, and
to a lesser extent
rev chile Med 2017; 145: 1099-1105
Investigation article
micellar conveyors *
Reason Col / Fos % Cholesterol Reason Col / Fos
0.10 89.5 0.13
0.20 59.7 0.08
Monounsaturated, lower total cholesterol and
LDL-cholesterol 31. Our studies agree with these results,
presenting a significant increase in both total plasma
cholesterol and LDL-cholesterol in animals that consumed
diets high in saturated fat. It has been suggested that the
increase in plasma cholesterol in individuals undergoing
high fat intake, occurs mainly in those with insulin
resistance, as they have lower intestinal absorption of
cholesterol 32.
Furthermore, it has been reported that levels of
HDL-cholesterol are increased in diets high long term
saturated or unsaturated fat, while low-fat diets,
significantly decreased levels of HDL cholesterol 29. This
effect is confirmed in our study, showing a significant
increase in HDL-cholesterol of 97% in the treated group.
This could explain why, in response to a high fat diet, a
likely drop is generated in hepatic uptake of HDL by
downregulating HDL receptor (Scavenger Receptor B1
-SRB1), preventing the reverse cholesterol transport 33.
Additionally, low-fat diets long-term showed an increase in
plasma triglyceride levels 29. Circulating triglycerides may
come from the intestinal fat absorption as chylomicrons
and chylomicron remnants, which can be highly
atherogenic 3. 4. In our study with short-term diet, we
observed no significant differences in plasma triglyceride
between the control group and the treaty, requiring further
research to assess the long-term effect of a high fat diet on
concentration and composition remaining lipoprotein.
Moreover, our results show no significant difference in
1103
Investigation article
glycemia levels between controls and treated animals.
Recent studies have also shown differences in response to
insulin resistance, between high and low fat diets 26,35.
The 3 main factors in the pathogenesis of cholelithiasis
are hypomotility of the gallbladder, bile cholesterol
supersaturation and crystallization of cholesterol.
Essentially, bile supersaturated with cholesterol results
primarily from an increase in biliary cholesterol secretion,
and / or a decrease in the secretion of bile acids, and to a
lesser extent of the decrease in the secretion of
phospholipids. In addition to these relative changes in
biliary lipids, the next step is important in the process of
formation of cholesterol gallstones, called crystallization,
because not always a supersaturated bile with cholesterol
crystals and subsequently form macroscopic calculations.
The crystallization process is cholesterol triggered by the
presence of vesicles which transport biliary cholesterol, but
they are thermodynamically unstable. One of the main risk
factors of cholelithiasis what constitutes excessive caloric
intake in the diet, where the resulting lipoprotein
cholesterol, which eventually returns to the liver, is
secreted into bile as bile acids or free cholesterol 36. In this
paper we study the role of dietary fat as etiological factor
of cholelithiasis. In our experimental model, we observed a
high fat diet, composed mainly of saturated fatty acids,
significantly increased the concentration of biliary
cholesterol without modifying the concentration of bile
phospholipids. This biliary cholesterol saturation caused
an increase in the concentration of cholesterol in the
vesicular carriers. Moreover, an increase in cholesterol /
phospholipid ratio vesicular transporters in the bile of
animals treated with high-fat diet was observed. These
vesicular transporters, high cholesterol, can be added and
merge, favoring the formation of cholesterol crystals 13.14. Probably Lipids 1992; 27: 526-32.
the saturated fatty acids in the diet caused a variation in
the composition of the bile phospholipids, which could
change the balance between the vesicular-micellar carriers
in bile, altering solubilization biliary cholesterol 37.
Conclude that a diet high in fatty acids, preferably
saturated, resulting in a
1104
fat intake and cholelithiasis - r. del Pozo et al
increased plasma levels of total cholesterol and
LDL-cholesterol, along with an increase in
HDL-cholesterol. Concomitant observed increase in the
concentration of bile cholesterol, and particularly the
increase in the formation of rich vesicular transporters in
cholesterol, indicates that eating a high-fat diet, preferably
saturated, could contribute to gallstone formation
cholesterol at an early stage. However, the mechanisms
involved have yet to be elucidated.
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