ABSTRACT
Globally, metabolic syndrome (MetS) has been on the rise and yet its risk factors are diverse in different populations. In most low income estates of major Kenyan cities and towns, roadside eating establishments reuse their both their cooking fats and oils in an attempt to minimize their businesses operating expenses. Some unscrupulous businessmen mix cooking fat with transformer oil to extend its cooking life: this over exposure of cooking fat to high temperatures and adulteration results in consumption of highly unstable oxidized and contaminated fats. The extended exposure to these abused fats poses a health risk and its physiological and toxic effects may have far reaching public health implications. The consumption of foods fried in these fats can possibly have an adverse effects to the body’s immune system and subsequently exacerbate long term conditions like HIV/AIDS, tuberculosis and cancers and other non-communicable diseases.
This study aimed to explore the effects of transformer oil-adulterated and reused cooking fats on the metabolic syndrome in male Sprague Dawley rats (Rattusnorvegicus).
Using a randomized experimental design, twenty four (24) weaned male Sprague Dawley rats were assigned to four (4) groups (n=6): ND, as control group I (fed a standard rat diet ad libitum), HFD, group II (fed 20% high fat diet ad libitum), RHFD, group III (fed 20% reused high fat diet) and group IV, RHFD + T.O, (fed a mixture of 20% reused high fat and transformer oil diet in the ratio of 3V:1V).To minimize on the expenses, measurements were undertaken at the end of the study period but not monitored over time. The groups mean body weights in grams were compared at the end of the study period as well as fasting blood sugar after 12 hours of fasting the experimental animals. After an intraperitoneal injection of Insulin, blood sugar was established at T0, T30, T60 and T120 and their mean of area under curve (AUC) used as a measure insulin tolerance. Venous blood was also collected at the end of the study period by a standard retro-orbital puncture for liver enzymes, lipid profile and osmotic fragility of the red blood cells assays. The study animals were then sacrificed and groups liver to body weight ratios determined. Finally the harvested liver samples were fixed in 10% neutral buffered formal saline (40% formaldehyde, 9% sodium chloride) and processed for Histopathology.
The experimental data were analyzed using One-way ANOVA followed by suitable post-hoc comparison performed using the statistical software Prism version 6.00 for Windows (GraphPad Software, La Jolla California USA). The significance level was set at p<0.05.
As compared to other study groups, group IV (RHFD+T.O diet) results had a significantly elevated osmotic fragility of the red blood cells of 108.8 ± 16.7 % at 0.34%NaCl, (p = 0.0043) as compared to the ND group, liver weight to body weight ratio was highest between RHFD + T.O and HFD groups (p = 0.0043), elevated liver enzyme ALT between RHFD + T.O and HFD groups (p = 0.0043), and a lipid disorder. Additionally, the same group had a significantly reduced body weight as compared to HFD, (p = 0.0087) with acute liver necrosis and significant portal inflammation of 2 according to the Non-alcoholic Steatohepatitis Clinical Research Network Histological grading system, indicating liver toxicity and bad health. Group III (20% RHFD) study animals were highly indicative of Non-alcoholic Steatohepatitis-inflammation of the liver concurrent with fat accumulation; the liver histology showed fat accumulation, inflammation, and fibrosis in different stages, this results were not well demonstrated in other study groups. Overall results further showed that all the groups on 20% high fat diet presented with differing levels of lipid disorders.
These findings demonstrate the hazardous effects of reused cooking fats and transformer oil mixtures and recommends for further studies and surveys on commercial use of cooking fats and oils in Kenya.
TABLE OF CONTENTS
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DEDICATION iii
ACKNOWLEDGEMENTS iv
LIST OF ABBREVIATIONS viii
LIST OF TABLES iv
LIST OF FIGURES X
LIST OF PLATES XI
ABSTRACT xiii
CHAPTER 1.0: INTRODUCTION
1.1 General introduction 1
1.2 Rationale 9
1.3.1 General objectives 9
1.3.2 Specific objectives 9
1.4 Research questions 10
CHAPTER 2.0 LITERATURE REVIEW
2.1 Saturated and unsaturated fats 11
2.2 Reused cooking fats. 18
2.3 Transformer oil 20
2.4 Digestion and Metabolism of fats. 22
2.5 Exogenous Metabolism of lipoprotein 25
2.6 Endogenous Metabolism of lipoprotein 26
2.7 Compositions and types of lipoproteins in human plasma 28
2.8 Cholesterol metabolism 30
2.9 Obesity and High fat diet 33
2.10 Diabetes and high fat diet 35
2.11 Blood Lipids, Cholesterol, Liver Enzymes and blood glucose levels. 39
CHAPTER 3.0: MATERIALS AND METHODS
3.1.0 : Materials 42
3.1.1 : Experimental animals 42
3.1.2 Neat cooking Fat 43
3.1.3 Reused cooking fat. 44
3.1.4 Transformer oil 45
3.1.5 Commercial rat pellets 46
3.1.6 Study design 47
3.2.0 Methods 48
3.2.1 Experimental design and diets 49
3.2.2 Preparation of diets 50
3.2.3 Body weights 50
3.2.4 Fasting blood sugar 50
3.2.5 Insulin tolerance test 51
3.2.6. Liver enzymes 51
3.2.7. Lipid profile 52
3.2.8. Percentages of liver to body weights ratio 53
3.2.9 Erythrocytes Osmotic fragility test. 53
3.2.10 Liver histology 54
3.3 Statistical methods 55
CHAPTER 4.0 RESULTS
4.1 Effects of the diet on mean body weight. 56
4.2 Fasting blood sugar. 57
4.3 Insulin tolerance test 58
4.4 Liver enzyme levels 59
4.4.1 Serum Aspartate Transaminases (AST) levels. 59
4.4.2 Serum Alanine Transaminases (ALT) levels 60
4.5 lipid profile 61
4.5.1 Serum Total Cholesterol 62
4.5.2 Serum High Density Lipoproteins (HDL) levels 63
4.5.3 Serum Triglycerides (TRIG) levels 64
4.5.4 Serum Low Lensity lipoproteins (LDL) levels. 65
4.6 Percentages of liver weights to body weights. 66
4.7 Osmotic fragility of erythrocytes 67
4.8 Histopathological evaluation of the stained liver cells 68
4.8.1 Physical appearances of the whole liver samples of the study 68
4.8.2 H & E histological appearance of the study liver samples… 66
CHAPTER 5.0: DISCUSSION
5.1 Changes in body weight 72
5.2 Percentage of liver weight to body weight 75
5.3 Osmotic fragility 75
5.4 Fasting blood sugar 73
5.5 Lipid profile 76
5.6 Liver enzymes 77
5.7 Histopathological examination 77
5.8 Insulin Tolerance Test 80
CHAPTER 6.0 CONCLUSION
6.1 Recommendation… 80
REFERENCES 85
LIST OF ABBREVIATIONS
IDF. -International Diabetes Federation.
MFMER. -Mayo Foundation for Medical Education and Research.
SREBP-CAP. -Sterol regulatory element-binding protein Cleavage-activating protein.
EDTA. -Ethylene diamine tetraacetic acid
DBPC. -Dibromochloro propane
Mdn. -Median
HFD. -High fat diet.
RHFD. -Reused high fat diet
RHFD+T.O. -Reused high fat diet with transformer oil.
ND. -Normal diet.
RBC. -Red blood cells
PCBs -Polychlorinated biphenyls.
ESR. -Erythrocytes sedimentation rate
TGs. -Triglycerides.
HDL. -High density lipoproteins.
LDL - Low density lipoprotein.
VLDL - Very low density lipoprotein
ALT -Alanine aminotransferase.
AST - Aspartate aminotransferase.
FFA - Free fatty acid.
IDL - Intermediate density lipoprotein.
MetS - Metabolic syndrome
ATSDRs - Agency for Toxic Substances and Disease Registry
TAGs - Tiacyglycerol
LIST OF TABLES
Table 1: Types and compositions of lipoproteins in human plasma. 30
Table 2. The nutritional information of rat pellets used in the study 47
Table 3: Histopathological evaluation of stained liver cells using the Non-alcoholic Steatohepatitis Clinical Research Network (NASH CRN) Histological grading system 73
LIST OF FIGURES
Figure1:Saturated and unsaturated fat acid molecule 12
Figure 2. Synthesis of a triglyceride 14
Figure 3. Biosynthesis of triacylglycerol molecule by dihydroxyacetone. 15
Figure 4. Synthesis of triacylglycerol molecule by the Monoacylglycerol. 16
Figure 5: Synthesis of triacylglycerol molecule via the diacylglycerol transacylases. 17
Figure 6: Chemical structure of a saturated triglyceride/triacylglycerol. 18
Figure 7: A simplified flow diagram of fat digestion. 23
Figure 8. Hydrolysis of Triacyglycerol by pancreatic lipase 23
Figure 9. Exogenous and endogenous lipid transport pathways., 27
Figure 10. A diagrammatic representation of the two pathways of cholesterol metabolism. 33
Figure 11: Study design for the study 48
Figure 12: Comparison of the mean body weight after 60 days in grams for the four treatment groups. 57
Figure 13: Comparison of fasting blood sugar in mmol/l after 60 days among the four groups. 58
Figure14: Bar graphs showing the glucose response during Insulin Tolerance Test among the four study groups., 59
Figure 15: Comparison of serum AST in I.U/l between the four groups 60
Figure 16: Comparisons of serum ALT levels between the four groups. t. 61
Figure 17. Comparisons of the serum total cholesterol between the four groups. 59
Figure 18: Comparisons of serum HDL cholesterol between the four groups 63
Figure 19: Comparisons of serum triglycerides between the four groups. 64
Figure 20: Comparisons of serum LDL cholesterol between the four groups 65
Figure 21: Comparison of liver weight/ body weight ratio between the four groups.,. 66
Figure 22: Comparisons of the osmotic fragility curves of erythrocytes between the four groups. 68
LIST OF PLATES
Plate 1: Photograph of some of the experimental rats before the start of the study 41
Plate 2: A photograph of a sample Frymate™ cooking fat. 42
Plate 3: Photograph of the used cooking fat. 43
Plate 4: A road side fish frying Kitchen 43
Plate 5: Photograph of a tin of the Gulf Transformer oil, UK 44
Plate 6: Group I whole liver sample of, (ND) 66
Plate 7: Group II whole liver sample, (HFD) 66
Plate 8: Group III whole liver sample, (RHFD)… 66
Plate 9: Group IV whole liver sample, (RHFD + T.O) 66
Plate 10: Group I: Liver histology of control group (ND),H&E X4. 67
Plate 11: Group II: Liver histology (HFD),H&E X4. 67
Plate 12: Group II: Liver histology (RHFD),H&E X4. 67
Plate 13: Group IV: Liver histology (RHFD + T.O), H&E X4. 67
Plate 14: Liver histology of group III study animals-RHFD (H&E X40), depicting features of microscopic cirrhosis 68
Plate 15: Liver histology of group III study animals, (RHFD) showing microscopic fibrous tissue, H & E X40. 68
Plate 16: Macro-nodular cirrhosis of reused high fat diet (RHFD) group III study animals 69
Plate 17: Micro nodular cirrhosis of reused high fat diet (RHFD) group III study animals. 69
Plate 18: Liver histology of group III study animals depicting features of NASH (Foci of Inflammation& hepatocellular ballooning. H&E X40) 69
Plate 19: Liver histology of group III study Animals showing more NASH features 69
CHAPTER 1.0
INTRODUCTION
1.1 General introduction
MetS is a collection of the most critical heart attack risk factors that include: diabetes and pre- diabetes, central obesity, hyperlipidemia and an elevated blood pressure(I.D.F.,2006).It’s approximated that 20-25% of the world’s adult population has the array of risk factors that may predispose them to MetS(Alberii et al., 2006).
The risk of having MetS is closely associated to obesity or abnormal weight gain that leads to a higher Basal Metabolic Index (BMI)(Cornier et al., 2008), other factors are; a sedentary life style, age, race, education status and gender.(Bankoski et al., 2011).However statistics of the syndrome in Kenya are not available, this may be due to consideration of the risk factors separately but not collectively as a syndrome.
A person is also determined as having the MetS if they have a central obesity plus two of the following four additional factors: elevated triglycerides (TGs), a depressed HDL-cholesterol, an elevated blood pressure or high than normal fasting plasma glucose level. Further studies have considered gender and ethnicity specific cut-off points for central obesity as determined by waist circumferences(International Diabetic Foundation, 2004).
The established guidelines of 2005 from the National Heart, Lung, and Blood Institute (America) and the American Heart Association consider any three of these traits in the same individual as the threshold for MetS: abdominal obesity established by a waist circumference of 102 cm (40 inches) or more in men and 88 cm (35 inches) or more in women, however for the Asian Americans, the cutoff values are ≥90 cm (35 in) in men or ≥80 cm (32 in) in women: a serum triglycerides of 3.9 mmol/L or above in both men and women: HDL cholesterol ≤1.04 mmol/ L in men and 1.3mmol/L or lower in women: blood pressure of 130/85 mmHg or more and a fasting blood glucose of 5.6 mmol/L and above.(Grundy et al., 2005).
It has been shown that subjectively healthy individuals may have biochemical abnormalities in keeping with the presence of MetS (Gami et al., 2007). In a study carried out in Kenya in 2017,a total of 528 participants found a prevalence of MetS was 25.6% (95% CI: 22.0%–29.5%). Among the surrogate markers of visceral adiposity, lipid accumulation product was the best predictor of MetS, while triglyceride was the best predictor among the lipid parameters for all participants. The optimal waist circumference cut-off for diagnosing MetS was 94 cm and 86 cm respectively for males and females. (Omuse et al., 2017). The study concluded that “The prevalence of MetS was high for a healthy population highlighting the fact that one can be physically healthy but have metabolic derangements indicative of an increased cardiovascular disease (CVD) risk. This is likely to result in an increase in the cases of CVD and type 2 diabetes in Kenya if interventions are not put in place to reverse this trend. We have also demonstrated the inappropriateness of the WC cut-off of 80 cm for black African women in Kenya when defining MetS and recommend adoption of 86 cm.”(Omuse et al., 2017).
A stratified sampling among University students in Kenya established that “1.9% of the participants met the criteria for diagnosis of metabolic syndrome according to Harmonized Joint Scientific Statement (HJSS) criteria. Among the elements, there was statistical difference in gender body mass index (BMI) and waist circumference. 11.8% of subjects had two metabolic syndrome components while 3.1% had three components while none of the subjects had all six components. Elevated triglycerides was the most prevalent defining component for metabolic syndrome. There is a statistically significant relationship between sedentary lifestyle and dietary habits as risk factors to metabolic syndrome.”(Mbugua et al., 2017)
In another cross-sectional study carried out at Riruta Health Centre (Kenya) in 2016, a population of 360 adults infected with HIVwere recruited and a questionnaire was employed to collect data on socio-demography. Their blood was analyzed for fasting glucose and lipid profile. The result indicated MetSpresent in 19.2% of the sampled population with prevalence higher among females participants than males. Obesity, lack of formal education, and family history of hypertension were associated with increased risk of metabolic syndrome while physical activity was associated with decreased risk. The study concluded that,“MetS is prevalent in this study population and the lack of formal education, obesity, physical inactivity and a family history of hypertension are associated with an elevated risk of MetS. Screening for risk factors, promotion of healthy lifestyle, and nutrition counselling should be offered routinely in HIV care and treatment clinics.”(Kiama et al., 2018).
A comparative study on the demographic characteristics i.e.abdominal circumference, weight, height, blood pressure, lipid profileand blood glucose) in Eldoret referral hospital (Kenya) on prevalence and correlates of metabolic syndrome and its components in 300 adults with psychotic disorders and 300 controlsfound out that patients with psychosis had a higher mean random blood glucose [5.23 vs 4.79, p = 0.003],higher triglycerides [1.98 vs 1.56, p<0.001], higher body mass index [5.23 vs 4.79, p = 0.001], lower high density lipoprotein [1.22 vs 1.32, p<0.001] andlarger waist circumference [89.23 vs 86.39, p = 0.009] . The risk of developingMetS were elevated with age and were reduced with female gender among those who were never married and among the widowed/separated/ divorced. Over a half of patients in this study were not receiving treatment for the various components of MetS. The study concluded that“metabolic syndrome and its components were more prevalent among patients with psychotic disorders than in controls; and a clear treatment gap for these disorders was evident. There is a need for efforts to ensure adequate screening and treatment for these physical disorders.”(Kwobah et al., 2021)
A cross-sectional baseline survey on gender variations in the pattern of socio-demographics relevant to MetS between Kenyan adults with central obesity at a Mission Hospital in Nairobi, Kenya, recorded a high occurrence of 87.2% MetS correlated with advanced age in males (p < 0.001) and females (p = 0.002). MetS was more likely among divorced/separated/widowed (p = 0.021) and high income males (p = 0.002) and females (p = 0.017). The unemployed males (p = 0.008) and the females with tertiary education (p = 0.019) unlikely to have MetS. Advanced age was more probable to lead to high blood pressure, fasting blood glucose and triglycerides (p < 0.001) had diminishedhigh density lipoproteins. The study did conclude that a high prevalence of MetS is highly associated with both social and gender differences among Kenyan adults with central obesity. The study finally recommended the need to look beyond behavioral and biological risks and focus with urgency on gender differences in dealing withMetS and cardiovascular diseases. (Okube et al., 2020).
It has also been indicated that these risk factors can also be an interplay of diets, genetics and gender. (Tian et al., 2017).The correlating data specifically for the Kenyan population is not available.
In Kenya, the prevalence rate of diabetes in adults is estimated at 2.0%(Guidelines-for- Screening-and-Management-of-Diabetic-Retinopathy-in-Kenya.Pdf.), however the World Health organization (WHO) has approximated the prevalence rate at 3.3% and projects a rise to 4.5% by 2025(Jones, 2013).This ballooning dilemma of diabetes in developing countries has been driven largely by a rise in obesity, according to WHO. In a 2012 WHO report, of the estimated 1.5 million global diabetes mortalities, greater than 80% occurred in low and middle income countries and W.H.O’s global activity on diabetes attributed 1% of the total deaths in Kenya directly to diabetes in the same year. (Jones, 2013).
The Kenya Directorate of Promotive Health Services outlines processed food, declined physical activities, Tobacco and alcohol abuse as important risk factors in diabetes(Mwenda et al., 2018).This has prompted the Kenya Diabetes Management and information Centre to embark on offering educative trainings on diabetes management and prevention, diabetes awareness, screening and monitoring: blood glucose, blood pressure, and Body Mass Index (BMI) among other activities in an attempt to scale down the prevalence of diabetes and its complications.
The Kenya Demographic and Health Survey (KDHS) estimated national prevalence of overweight and obesity for women of 15-49 years of age at 23% with a disproportionately higher rate in urban centers.(Mbochi et al., 2012) The 2018 Health Sector Performance Review Report for 2016/2018 puts Hypertension as the leading Non Communicable Disease (NCDs) diagnosed during outpatient visits. The report also highlights the number of new cases of hypertension as three times the number of Diabetes as reported in outpatient department. These major NCDsare mostly brought about by lifestyle habits that include the type and quantity of cooking fats and oils used in preparation of meals.
Most solid fats are higher in saturated and/or trans-fats and lower in monounsaturated or polyunsaturated fats (PUFAs) than oils. Tran-fats and saturated fats are known to increase the low density lipoproteins (LDL) in the blood which eventually elevates the risk for heart disease. (Hunter, 2005).
In Kenya some of the most popular brands of cooking fats include; Fry mate (Pwani oil products Ltd), Mallo and Chipsy (Bidco oil Refineries), and Kasuku (Kapa oil Refeniries). (Best Cooking Oil and Fats in Kenya and Their Available Sizes - Info KE, 2021). This study used FryMate cooking fat due to its popularity by then as asserted by the fish vendor with whom i collaborated with in recycling of the cooking fat.
The high prevalence of diabetes in social and economically disadvantaged urban populations has been capturedby two studies in two major slums in Kenya (Korogocho and Viwandani).The studies indicated prevalence of diabetes in this impoverished population as moderately high.(Van de Vijver et al., 2013). The same study of this urban population revealed obesity as an important risk factor for raised blood glucose levels especially among women(Van de Vijver et al., 2013).
In another study in the same urban slum areas, it was found that the rates of treatment, awareness, and control were low and obesity was single most important risk factor for a raised blood glucose especially in women. (Oti et al., 2013). The study further noted that once individuals are aware of their hypertensive status, most will seek remedial measures. “Overall, there is urgent need to implement strategies that improve on prevention, detection, and affordable access to effective treatment in these populations.”(Van de Vijver et al., 2013).
In a 2012 cross sectional study among some five urban social economic clusters (upper, lower upper, middle and lower) of Langata constituency in Nairobi, Kenya, the prevalence of MetS was found to be at around 34.6 %.(Kaduka et al., 2012), and the most distinctive characteristics of MetSwere: elevated blood pressure, a higher than normal waist circumference, and lower than normal HDL cholesterol. This study however noted that elevated fasting blood glucose and triacylglycerol (TAGs) were less frequent (Kaduka et al., 2012).The study further noted that the main risk factors linked to prevalence of MetS in these urban populations were increase in age, a lower measure of their education level and income occupation: all of which inform their choice of diets (Kaduka et al., 2012)
In developing countries, there has been a rapid nutrition transition from a high fiber, high spices and whole grain diet to a highly processed caloric diet coinciding with increases in obesity, MetS, and type 2 DM(Misra et al., 2010). Available data shows an increase in supply and use of cooking fats that is consistent with high intake of these saturated fats in these developing countries (Misraet al., 2010). Most likely these modern diets together with a sedentary lifestyle have compounded the risk factors for MetS.
Mixtures of Transformer oil and standard cooking fat have been reported to be used in deep- frying foods in informal food kiosks in a number of countries including Kenya, Zambia, Zimbabwe, Tanzania, Malawi, and Bangladesh. Deep fried food stuffs are sold almost anywhere and most clients unknowingly buy these foodstuffs for consumption. (The Post Newspaper, Zambia. Monday 29 March 2010).In Kenya transformer oil siphoning for cooking among other uses was estimated to cost the Power firm Ksh 60 million a year. (The Daily Nation Newspaper. Thursday 22 November, 2012). In a 2007 report entitled: “challenges of vandalism on power equipment” by Engineer Joseph K. Njoroge, Managing Director, The Kenya Power and Lighting Company Limited, he notes that among many other uses of vandalized Transformer oil, it is mixed with vegetable cooking oil and sold as cooking oil(Njoroge, 2007). The main reason behind this dangerous business behavior is a longer frying period and maximization of profit.
Overall, the recommended remedies for MetS are lifestyle changes that include losing excess weight, eating a healthy diet, being physically active, stopping smoking in addition to medications for high blood pressure, cholesterol and elevated blood sugar.
Conclusively, prevention and any meaningful control strategies that target possible modifiable risk factors for diabetes and increase access to affordable treatment in any disadvantaged settings are urgently needed(Kaduka et al., 2012).
1.2 Rationale
This study hopes to contribute to the general understanding how reused cooking fats and transformer oil may be some of the precipitating factors in Metabolic Syndrome,
1.3 Objectives
1.3.1 General objectives
To investigate mixtures of reused cooking fat and transformer oil as risk factors in development of Metabolic Syndrome in male Sprague Dawley rats(Rattusnorvegicus).
1.3.2 Specific objectives
1. To compare lipid profiles of experimental animals fed different lipid dominated diets.
2. To compare levels of liver function enzymes in the study animals fed different lipid dominated diets.
3. To compare fasting and random blood glucose levels in the study animals fed different lipid dominated diets.
4. To compare liver to body weight ratios and overall body weights in study animals fed different lipid dominated diets.
5. To compare and assess the form and structure by liver histology of the different study animals.
1.4 Research questions
i. How do mixtures of reused cooking fat and transformer oil affect blood lipid profiles in male Sprague Dawley rats?
ii. How do mixtures of reused cooking fats and transformer oil affect critical liver enzyme levels in male Sprague Dawley rats?
iii. How do mixtures of reused cooking fats and transformer oil influence the body weight of the study animal’s livers?
iv. How do mixtures of cooking fats and transformer oil affect blood sugar levels in the study animals?
v. How do mixtures of cooking fats and transformer oil affect the form and structure of the study animals?
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