1) Beakers
2) cornical flask
3) Petric dish
4) Crucible
5) Hot plate
6) Measuring cylinder
7) Kyedhal setup
8) Fume Cupboard
9) Funnel
10) Digestion bottle
11) Heating Mantle
12) Filter paper
13) Weighing balance
14) Oven
15) Spatula
16) Furnace
3.1.2 MATERIALS FOR HISTOLOGICAL AND HEMATOLOGICAL PROCESSES
1) Microtome machine
2) Water bath
3) Glass slide
4) Oven
5) Glass staining jar
6) Wooden block
7) Ice block
8) Microscope
9) Round bottom flask
10) Centrifuge model 90
11) Spectrophotometer
12) Test tubes
13) Cuvelte
14) Electronic balance
15) Cholesterol kit
16) High density lipoprotein kit
17) Distilled water
18) Micropipette
3.1.3 CHEMICALS FOR FOOD ANALYSIS:
1) Petroleum ether
2) Copper Sulphate
3) Sodium Sulphate
4) Concentrated H2SO4 (Sulphuric acid)
5) Sodium Hydroxide (NaoH)
3.1.4 CHEMICALS FOR HISTOLOGICAL AND
HEMATOLOGICAL ANALYSIS
1 Alcohol
2 Xylene
3 Paraffin wax
4 Haematoxylin
5 Eosin
6 Formol saline
7 1% Acid alcohol
8 Distilled water
9 Leishman’s stain
3.2 EXPERIMENTAL ANIMAL
20 apparently healthy albino wistar rats were used for the study. The rats were purchased from the animal house of the University of Nigeria, Nsukka. The animals were maintained at temperature of 30 degrees to 36 degrees Celsius and fed with growers mash manufactured by Grand cereal Ltd, a subsidiary of UAC of Nigeria Plc Jos Plateau and was purchased at #10 OHAOFIA Street, Abakaliki, Ebonyi state. The rats weighed 20-50g and were acclimatized at the animal house of the College of Health Science, Ebonyi state University, for a period of 2 weeks.
The animals were randomly assigned to 2 groups (African dietary group and European dietary group) with 10 animals in each group. The African dietary group served as the control group while the European dietary group served as the experimental group. The animals were cared for in compliance with the applicable guidelines
3.3 EXPERIMENTAL PROCEDURE
All the animals in each group were allowed food and water for 12 weeks. The European group of experimental animals were fed with a compounded feed made from fried meat and egg yolk. The animals received 50grams of food everyday for 12weeks, blood was collected through ocular puncture after which the animals were sacrificed using cervical dislocation, the animals were dissected and the whole heart was cut and fixed for histological analysis.
3.4 DIETARY VALUES
The dietary values of the animals feed is made up of approximately the following
Carbohydrate = 82.5%
Protein = 4.2%
Fats = 3.8%
Moisture = 6.5%
Fibre = 1.5%
The compounded feed to mimic European diet consisted of fried meat and egg yolk; and their dietary values is made up of approximately the following;
Carbohydrate = 24.83%
Protein = 11.37%
Fats = 42.5%
Fibre = 1.8%
Ash = 2.5%
This dietary values was analyzed in the food chemistry and microbiology laboratory of the Department of Food Science and Technology of the Faculty of Agriculture, Ebonyi State University, Abakaliki.
3.5 ANIMAL FEEDING
The animals in each group were fed as follows’
Group1(control) was fed on normal animals feed which mimic African diet rich in carbohydrate and low in protein and fats.
Group2(Experimenal group) was fed on a compounded feed containing 40grams of meat and egg yolk and 10grams of normal feed to mimic European diet.
All the rats were fed 50grams of their feed every day.
3.6 DETERMINATION OF THE DIFFERENTIAL WHITE BLOOD CELL COUNT
Blood collection analysis
On the 12th week, each rat was anaesthetized with chloroform. About 5mls of blood was bled by ocular puncture using microhaematoent capillary tube into ETDA bottles and labeled. Within 2 hours, blood films was made and stained with Leishman’s stain and differential leucocyte counts conducted randomly on 200 leucocytes in each slide under the microscope was counted.
3.7 DETERMINATION OF LIPID PROFILE
The following lipid profile were determined
3.7.1 DETERMINATION OF TOTAL CHOLESTEROL.
The method of Trinder (1969) was used for its determination
Principle: The cholesterol is determined after enzymatic hydrolyses and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4- animoantipyrine in the presence of phenol and peroxidase.
Cholesterol ester + H20 cholesterol esterase cholesterol + fatty acid
Cholesterol + O2 cholesterol oxidase cholestene-3-one + H2O2
2H2O + Phenol + 4-aminoantipyrine peroxidase Quinoneimine+ 4H2O.
Procedure: cholesterol Randox test Kits were used. The kits have the following as the reagent composition. R1 Reagent (4-aminoantipyrine, Phenol, Peroxidase, cholesterol esterase, cholesterol oxidase, pipes Buffers) and calculated standard. Testtubes labeled reagent blank and sample were set up into the test tubes labeled reagent black, 20.0ul of distilled water and 2000.0ul of reagent were added, while 20.0ul of sample (plasma) and 2000.0ul of reagent were added to the test tube labeled sample. The content of each test tubes were mixed differently and incubated for 5 minutes at 370C. the absorbance of the sample (A sample) against the reagent blank was measured at 546mm using spectrophotometer within 60minutes.
The cholesterol level was determined using the formular
∆A sample X concentration of standard
∆A standard
3.7.2 DETERMINATION OF HIGH DENSITY LIPOPROTEIN
The method of lope- veriella et al, (1977) was used in the determination.
Principe: Low density lipoprotein (LDL and VLDL) and chylomicron fractions are precipitated qualitatively by the addition of phosphotungstic acid in the presence of magnesium ions. After centrifugation, the cholesterol concentration in the high density lipoprotein fraction which remains in the supernatant is determined.
Procedure:
High density lipoprotein Randox test Kits were used. The kits have the following reagent composition. R1 (Phosphotungstic acid, magnesium chloride).
Precipitation
Into a centrifuge tube, 500.0ul of sample and 1000.0ul of precipitant (R1) were added. These were gently mixed and allowed to sit for 10 minutes at room temperature. It was then centrifuged at 4000g. the clear supernatant was then separated within 2 hours and cholesterol content determined by the CHOD-PAP method.
Cholesterol CHOD-PAP ASSAY.
Test tube labeled reagent blank and sample were set up in the test tube labeled reagent blank, 200ul and 2000ul of reagent were added, while 200ul of supernatant and 2000ul of reagent were added to the test tube labeled sample the content of each test tube were mixed and incubated for 5 minutes at 370C. The absorbance of the sample (A sample) against the reagent blank at 500nm was measured within 60minuts.
The values of the high density lipoprotein cholesterol was determined using the formulae;
∆A sample X concentration of standard
∆A standard
3.8 HISTOLOGICAL PROCESS OF THE HEART
The whole heart was cut and fixed. Thin sections of the aorta were cut at 5um and stained using H and E staining method
The following steps were taken for tissues processing;
Fixation: The tissue were fixed in 10% fromol saline for one week.
Dehydration: Was carried out through ascending grades of alcohol (50%, 70%, 90%, and absolute alcohol) for 45 minutes respectively.
Clearing: This was done in three changes of xylene for 45 minutes each.
Impregnation: This was done in a hot oven in three changes of paraffin wax for 30 minutes each
Embedding: The impregnated tissues were embedded using embedding mould and allowed to solidify.
Trimming: The paraffin block was trimmed with blunt microtome knife and sticked on the wooden block for microtomy
Staining: This was carried out using a cleanslide, staining jar and the following procedures were undertaken.
A) Paraffin sections were dewaxed through 2 changes of xylene each for 3minutes.
B) Sections were rehydrated through descending grades of alcohol in 2 changes for 1 minute each.
C: Sections were rinsed in running tapwater for 5 seconds
D) Section were stained with hermotoxylin for 15mins
E) Sections were rinsed in tap water for 5 seconds.
F) Sections were differentiated in 1 mile of acid alcohol for 1 second.
G) Sections were blued in running tap water for 10 minutes
H) Sections were counterstained with eosion for 5 minutes
I) sections were rinsed in water for 2 times
J) Sections were dehydrated by passing through 90% alcohol for 15 seconds and absolute alcohol for 15 seconds.
K) Sections were transferred to xylene
L) sections were mounted in DPX and covered with coverslips.
M) Dried in the Oven.
Result Nuclei – BLUE
Cytoplasm – pink red
3.9 STATISTICAL ANALYSIS
The results of the present study were expressed as mean±SD, statistical analysis for significant differences between control group and European dietary group was done using student T test (P≤0.001).
4.0 RESULT
The results demonstrates a significant change/difference in the histology of the heart of the wistar rats fed on European diet compared to the rats fed on the African (control) dietary group. These significant difference in the histology of the heart of rats in the European dietary fed group and African dietary fed group are shown in fig 1 to 10
From fig 1 to 5, The result of the photomicrograph of the African (control) dietary fed rats revealed a central aorta surrounded by the smooth muscles of the layers of the aorta with the surrounding cardiac muscles.
In fig 6: The rats fed with European diet showed hypertrophy of the smooth muscles of the layers of the aorta (tunica intima, tunica media and tunica adventitia) while the lumen increased in volume compared with the control African dietary fed rat.
The Photomicrograph of the European dietary fed rat in fig 7 shows a significant deposition of fat on the smooth muscles of the layers of the aorta compared with photomicrograph of the African dietary fed rats
In fig 8 rat fed with the European diet, shows a significant focal area of fat deposition on the smooth muscles of the layers of the aorta compared with the control African dietary fed rats.
Figure 9: also revealed an extensive deposition of fat within the smooth muscles of the layers of the aorta of the rat fed with European diet compared with the control African dietary fed rats after a feeding period of 12 weeks.
In the rat fed with the European diet, the result of the photomicrography also demonstrates hypertrophy of the smooth muscles of the layers of the aorta as shown in fig 10.
The rats fed in the European dietary group also had a significantly higher percentage neutrophil count and low percentage leucocyte count when compared with the rats fed on African (control) dietary group after a 12 week feeding period) as shown in the table 1.
The plasma total cholesterol level of the European dietary group significantly increased when compared with control (African) dietary group after a 12 week feeding period as shown in table1. On the contrary, there was no significant difference in the high density lipoprotein (HDL) level between the European dietary group and the African (Control) dietary group after a 12 week feeding period as shown in the table 1.
DISCUSSION
Several studies have shown that the main difference between traditional African diet and European diet is the absence of high level of cholesterol and saturated fatty acids in the African diets (Green et al, 1978, Fredickson, 1974). Various studies have also indicated that high serum level of cholesterol is positively correlated to atherosclerosis and increased risk of coronary heart disease (Nwanjo et al, 2007, Meijer et al, 1996).
Egg and meat are one of the most concentrated source of cholesterol in the diet. Eggs provide approximately 35% while meat provide 44.6% of the dietary cholesterol in the diet according to Densie Webb, 2011. Research has also shown that for each 1% rise in serum total cholesterol, the risks of coronary heart disease increases by about 2%. It also showed that subjects with the highest cholesterol intake experienced a 38% increase risk of coronary heart disease compared with those in the lowest intake group (Shekelle et al, 1989).
The present study demonstrates a significant increase in the percentage neutrophil count of the European dietary fed rats after a 12 weeks period (P≤0.001) compared with the control African dietary group. The percentage lymphocyte counts of the control African dietary group increased significantly after the 12 weeks feeding period (P≤0.001). The weight in grams of the wistar rats fed in European dietary group also increased significantly compared to those on the control African dietary group. These agreed with the work of Oji, et al, 2011 that reported a significantly higher percentage neutrophil count and a lower percentage lymphocyte count of rats fed on high saturated fat, high protein and low carbohydrate (European diet) when compared with the control African dietary group fed on high carbohydrate, low saturated fat and protein diets. This also agreed with the work of Ezeilo, 1972 which reported that neutropenia in Africans is dietary in origin and if the diets of Africans changes to European type (high saturated fatty acid and protein), the frequency of neutropenia will drop noticeably and if Africans had European diets form infancy, Neutropenia would probably disappear. Meijer et al, 1996 also reported that elevated (high) dietary cholesterol increases blood total leucocyte (Higher neutrophil counts).
The present study also demonstrates significant increase in the total cholesterol level in the European dietary fed group as compared with the control African dietary fed group. This agrees with the work of Oji et al, 2011 that reported a significantly higher total cholesterol, low density lipoprotein and triglyceride of the rats fed on high saturated fat, high protein and low carbohydrate (European diet) when compared with the control African dietary group fed on high carbohydrate, low saturated fats and protein diets.
There was NO significant difference in the high density lipoprotein (HDL) level between the European dietary fed group and the African control dietary fed group of the albino wistar rats. This agrees with the works of Abdelhalim et al, 1994 who reported that the high density lipoprotein was not significantly different after feeding rabbits with cholesterol for 15 weeks compared with the control group.
As observed in the histological slide, there are hypertrophy of the smooth muscles of the layers of the aorta, focal areas of fat deposition, extensive fat deposition on the smooth muscles of the aorta. This agrees with the work of Meijer et al, 1996 that hypothesized that high blood cholesterol influences cardiovascular diseases. The low HDL levels are associated with an elevated blood viscosity and this rheological abnormality contributes to cardiovascular risks (Thomas et al, 1999). It has also been reported that serum hypercholesterolemia accelerates atherogenesis and contributes to symptomatic atherosclerosis by increasing blood viscosity and disturbing the mechanical fragility of atherosclerotic plaque making them vulnerable to rupture and thrombosis (Gregory, 1999).
CONCLUSION AND RECOMMENDATION
The results of the present study suggest that the European diets (high cholesterol and saturated fat) increases percentage neutrophil count compared to African diets which have been reported to be responsible for Neutropenia in Africans.
The elevated total cholesterol may be associated with the pathophysiology of the atherosclerotic process in the European dietary group which is a predisposing factor for cardiovascular diseases.
Therefore, there is need to moderate the intake of dietary cholesterol and yet maintain high neutrophil count to avoid the risk of cardiovascular diseases. There is also need to maintain an active lifestyle by engaging in exercises in other to expend high amount of energy and reduce blood cholesterol level.
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