Patterns of Some Immunological Proteins and Oxidative Indices in Normal and High Body Mass Index Wistar Rats Fed Coffee and Aspartame

This study evaluated the effect of oral intake of coffee and aspartame on the oxidative status and some cytokines levels in varied body mass index Wistar rats. Ninety-six male Wistar rats were allotted into 2 sets of four groups respectively, comprised of twelve rats with average weights of 147.15 ± 4.96 g (normal body mass index (N-BMI) and 269.42 ± 6.14 g (high body mass index (H-BMI). The rats were administered coffee and aspartame such that: Groups A and B rats were respectively N-BMI and H-BMI, received distilled water; Groups C and D rats were respectively N-BMI and H-BMI, received coffee; while E and F rats were respectively N-BMI and H-BMI, received aspartame; and G and H rats were respectively N-BMI and H-BMI, received aspartame and coffee. Six rats from each group were sacrificed after 6 weeks of administration, while the remaining six rats were sacrificed after 12 weeks of administration. The concentrations of nitric oxide (NO), superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA), catalase, protein carbonyl (PC), protein thiol (PT), S-glutathionylated protein (S-GP), C-reactive protein (CRP), interleukin 6 (IL6), interleukin 1β (IL-1β), vascular cell adhesion molecule (VCAM-1), monocyte chemotactic protein-1 (MCP-1), oxidized low density lipoprotein cholesterol (ox-LDLC), paraoxonase (PON-1) and total immunoglobulin (IGT) were determined in the serum, plasma and tissues homogenate as applicable. The histo-pathological analysis was performed on the liver. The results of the study indicated decreases (p<0.05) in NO, MDA, PC, S-GP in N-BMI rats administered coffee and aspartame both at 6 weeks and 12 weeks, while increases were obtained in GSH and SOD in the coffee administered N-BMI rats. Marked increases were recorded in NO, IL-6, IL-1β, CRP, MCP-1, VCAM-1, ox-LDLC and IGT both at week 6 and week 12 in no duration dependent manners (p<0.05) across the groups. Photomicrographs of the liver of H-BMI and aspartame N-BMI rats indicated varied alteration in the histo-architecture of hepatocytes. The present data indicated that the sole administration of coffee ameliorated some inflammatory perturbations recorded in the H-BMI rats. However, the trend was not sustained in the N-BMI and H-BMI rats that received aspartame, rather the exacerbation of the perturbations were recorded. Consequently, it is recommended that long-term consumption of aspartame might be correlated with oxidative stress and inflammatory disorders, and BMI is directly implicated in the aforementioned conditions. antioxidative high-density lipoprotein in prevent


Introduction
Body fatness has been an important psychosocial issue among humans since time immemorial. Traditionally, a person's fatness can be defined at a personal level, as well as at a societal level.
Thus, every individual has his / her own perception of how fat he / she could be. At the societal level, although it is poorly described or quantified, but there also is a degree of fatness beyond which a person generally is considered to be unacceptably fat; that is, there is an ill-defined threshold at which a person is labeled as being "too fat" or "obese." The social consequences of being "obese" are severe [1]. Discrimination begins in childhood and results in serious emotional scars. Societal discrimination limits career choices, and indeed many career paths are closed to those considered to be obese. Also, societal stigmatization often impairs a person's ability to express his/her intellectual and other talents; that is, they are often termed underachievers [2]. Clinically, obesity is an epidemic in the present world that has caused a lot of health concerns. Obesity raises the risk of early mortality and other chronic diseases and is associated with increased health care costs, placing a large financial burden on the populace [3]. In fact, it was considered that six out of 10 adults are considered to be overweight, which is determined by the body mass index. Body Mass Index (BMI) is a measurement of the weight with respect to the height of an individual. It is more of an indicator than the direct measurement of a person's total body fat. Thus, as the BMI score increases, so does a person's total body fat [4].
The degree of fatness has various interpretations and acceptance in different cultures is not clear. However, it may have depended on the availability of a reliable food supply and the effort required in obtaining it. This concept has moderated but still influences people's views of being smart, healthy, mentally alert, as well as the good eating habits. All these factors have necessitated the need to control or be conscious of individual's BMI to avoid the negative societal and health issues associated with being overweight. Plausibly, this could be the reason for the increase in the consumption more of fibers and low-calorie diets, the use of energy mobilizers, such as coffee, caffeinated drinks or chocolate, the preferred use of no calorie sweeteners instead of sugar, moderate consumption of grape wines, herbal preparations etc., so as to control body fat. Therefore, the consumption of caffeine, either in coffee or carbonated cola drinks (diet coke), as well as the use of aspartame as sweetener by individuals that seek to control the BMI can't be overemphasized.
Coffee is one of the most widely used non-alcoholic beverages in developing and developed countries. The coffee tree belongs to the Rubiaceae family, genus Coffea. About 80 coffee species have been identified worldwide, but only two are economically important [5].
The two economically important species are Coffea arabica, also known as Arabica coffee and Coffea canephora or Robusta coffee [6,7]. Although, coffee is made up of several components, caffeine is the most abundant constituent of coffee. Other common dietary sources of caffeine are tea, diet coke, cola soft drink, chocolates and so on. Caffeine exerts a variety of stimulatory effects upon the central nervous system, and it is probably the most widely used psychoactive substance. It also increases the respiratory rate, causes bronchi dilation, and stimulates lipolysis [8]. It is well documented that caffeine is a psychoactive drug associated with increased cognition and alertness. In fact, the stimulating effect of caffeine is the core pharmacological component of coffee. Thus, coffee is consumed largely by many in other to be active and it is part of everyone's daily drink.
Aspartame, L-aspartyl-L-phenylalanine methyl ester, is a white powder with no odor that is extremely sweet to taste. It is about 200 times sweeter than sugar thus a very small amount provides the same sweet taste as sugar. Aspartame is a non-nutritive intense artificial sweetener used by the general public as an alternative to sugar [9,10]. Since regulatory approval about 20 years ago, the high-intensity sweetener, aspartame, is consumed in more than 6000 products by hundreds of millions of people around the world who want to enjoy the sweet taste of sugar without all the calories [11]. It is available in various food products such as beverages, dairy products, cereals, chewing gums, canned fruits, energy drink, zero coke, desserts, confections etc. Metabolism of aspartame generates approximately 50 % phenylalanine, 40 % aspartic acid and 10 % methanol respectively [12]. Phenylalanine and aspartic acid are both amino acids which are found in natural proteins and under normal circumstances are beneficial, if not essential, for health [13]. A small amount of aspartame can relatively increase plasma methanol levels. Methanol is being increasingly recognized as a substance that damages the liver cells where it is oxidized to formaldehyde and later to formate [14]. Although several studies have reported the individual effects of coffee and aspartame on the health statuses [15][16][17][18][19][20], but no literatures are available on caffeine or coffee consumption in combination with aspartame on the different biochemical variables in individuals with either high BMI or normal BMI. Relatively, there is dearth of information on the relationship between the BMI and the use of caffeine or coffee, and aspartame coadministration as found in diet coke, energy drinks etc. Hence, this study was undertaken to evaluate some health implications of the consumption of coffee and aspartame as a sweetener in normal and overweight consumers. We, therefore, investigated the oxidative status and patterns in some immunological proteins following the sub-chronic administration of coffee and aspartame singly or in combination, in normal and overweight male Wistar rats.

Animal Handling, Administration of Coffee and Aspartame
The rats were kept in compartmentalized cages and grouped into eight, comprising of twelve animals each. They were allowed access to standard pellets and distilled water. The cages were cleaned every day and the animals were kept under condition of uniform humidity and temperature on a 12-hour light -dark cycle. The animals were allowed to acclimatize for two weeks, during which they were weighed and properly observed for any changes in their behavior. Of the ninety-six rats, forty-eight were fed standard rat pellet, while the other forty eight were fed atherogenic diet for the period of six weeks to achieve mean weights of 147.15 ± 4.96 g (normal body mass index (N-BMI) and 269.42 ± 6.14 g (high body mass index (H-BMI). respectively. Coffee and aspartame were administered orally at 35 and 60 mg/kg body weight respectively to the rats such that: Groups A and B rats respectively were N-BMI and H-BMI and received distilled water. Groups C and D rats were respectively N-BMI and H-BMI and received coffee, while E and F rats were respectively N-BMI and H-BMI and received aspartame.
Rats in groups G and H were respectively N-BMI and H-BMI and received aspartame and coffee concurrently. After 6 weeks of administration, six rats from each group were sacrificed, while the remaining six rats were sacrificed after 12 weeks of administration.
The feed intake and body weights of the rats were monitored during the administration.

Collection of Plasma and Serum
The rats were fasted overnight and anaesthetized using diethyl ether. Blood was collected via cardiac puncture and the plasma and serum were prepared for each drawn blood. Organs of interest, such as the liver and brain were harvested immediately, cleansed of blood and rinsed with normal saline solution and the weight recorded. The organs were minced, and a small part was fixed in 10 % formasaline for histopathology examination.

Determination of Biochemical Parameters
Oxidative Status: The activities of superoxide dismutase and catalase were determined as instructed in the reagent kit manuals, as well as the concentrations of reduced glutathione, malondialdehyde, nitric oxide, protein carbonyl and total protein thiol serum or plasma and tissues homogenate as applicable.

Immunological
Proteins: The

Oxidative Stress Indices
The trends obtained in the oxidative stress indices determined in this study were presented in Table 1. Marked reductions (p<0.05) were recorded in the concentration of reduced glutathione in the liver of rats across the groups in a manner not dependent on the duration of administration, except in groups C and D rats (p>0.05).
Glutathione peroxidase concentrations were not altered (p<0.05) in the livers of rats in groups B and C at both weeks, and groups D and Hat six weeks only. The production of nitric oxide in the plasma and malondialdehyde concentrations in the liver of rats increased (p<0.05) across the groups in a duration dependent manner, except in rats in group B, while malondialdehyde concentrations in the blood increased (p<0.05) consistently across all the groups Table   1. The activities of superoxide dismutase decreased (p<0.05) in the liver and plasma of the rats across the groups, which were dependent on the duration. In a similar manner, catalase activities in the liver were diminished across the groups (p<0.05), except in group B (p>0.05).

Inflammatory Indices
The results obtained in the inflammatory indices determined in this study were depicted in the

Immunological Parameters
Depicted in Table 3 were the patterns recorded in the immunological parameters assayed in our study.        There is an area of edema noted (black arrow), the sinusoid appear mildly dilated with infiltration of inflammatory cells (slender arrow), the hepatocytes show cytomorphology with necrosis (blue arrow). X=6 weeks and Y=12weeks.

Discussion
Nitric oxide is important for the relaxation of smooth muscles to enhance the flow of blood and increase general cell health.
The biological activity of nitric oxide may be modified by oxygenderived reactive species, such as anion superoxide (O2•), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH•), contributing to regulate the vascular tone [22]. In this study, the increases recorded in the production of nitric oxide (NO•) in the rats with normal-and high-body mass index (N-BMI and H-BMI) administered with aspartame and coffee, either singly or in combination indicated predisposition to the generation of free radicals Table 1 co-substrate for peroxide detoxification by glutathione peroxides [25]. The patterns in the glutathione peroxidase activities in the liver of rat supported the reported depletion of GSH in the liver Table 1. Glutathione peroxidase is a major enzyme that in involved in the neutralization of free radicals generated during cellular and or, xenobiotic metabolism, and its activity is dependent on the concentration of GSH. The reductions in GSH and glutathione peroxidase indicated a burden / an overwhelming effect on the endogenous antioxidant systems. This is supported by the reported patterns in the superoxide dismutase (SOD) and catalase (CAT) activities in the livers of rats, which were amplified by the duration of administration and the BMI. In addition, the reductions in antioxidant indices (GSH, glutathione peroxidase, CAT and SOD) in the liver of rats administered aspartame and the increases when co administered with coffee, implied that coffee had ameliorative capacity on the antioxidant systems Table 1. The result of this study underlined the antioxidant activity of coffee and oxidative capabilities of aspartame. An earlier report confirmed that methanol administration could decrease the enzymatic antioxidants (SOD and CAT) in the lymphoid organs [26].
The decline in the activities of these enzymes might be due to their inactivation caused by excess reactive oxygen species (ROS) production [27]. Normally, the antioxidant enzyme, catalase protects SOD against inactivation by H 2 O 2 . Reciprocally, SOD protects catalase against superoxide anion. However, overload of free radical could upset these regulations. Furthermore, the decrease in SOD and CAT activities might be due to the formation of formaldehyde from the methanol. Previous studies have showed that aspartame metabolism produces methanol, which is oxidized to formaldehyde and in turn increase free radical production that could lead to decreases in SOD and CAT activities in the liver [28].  Table 2.
Oxidized low density lipoprotein cholesterol contains unoxidized and oxidized fatty acid derivatives both in the ester and free forms, their decomposition products, cholesterol and its oxidized products, proteins with oxidized amino acids and cross-links, and polypeptides with varying extents of covalent modification with lipid oxidation products, and many others [31]. Although, there were no scientific associations between ox-LDL-C concentrations and the severity of inflammation, as the risk of ox-LDL-C formation did not change after adjustment of some inflammatory markers (Wu, 2006). However, oxidative modification of LDL-C promoted fatty-streak formation, the early lesion of atherosclerosis. This is because ox-LDL-C was shown to induce production of monocyte chemotactic protein-1 (MCP-1) and monocyte-colony stimulating factor and lead to inflammatory response in vascular cells [32].
The reported decreases in the concentrations of protein thiol and increases in protein carbonyl and S-glutathionylated protein in the liver in rats administered coffee and aspartame Table 2 indicated that aspartame had oxidative capabilities and coffee had sparing or little ameliorative potential when co-administered at the doses.
S-glutathionylation is the reversible formation of protein mixed disulfides between cysteine residues and GSH (post-translational modification that is important to many biological processes). It is also an important form of post-translational modification that occurs during oxidative stress as well as under normal physiological conditions. Reversible protein thiol oxidation has been demonstrated in proteins with range of functions including signal transduction, ion transport, contractility and protein metabolism [33]. The sulfhydryl groups in protein thiol are very susceptible to oxidative damage and are often low in-patient suffering from disease and infection.
Protein carbonyl content in blood and tissues is a reliable indicator of protein oxidation [34]. The trend obtained in protein carbonyl was consistent with increases observed in the MDA level of aspartame administered rats and H-BMI rats (Table 1 and 2 respectively). In addition, H-BMI could have predisposed the rats to  Table 3 gave credence to the increased need of monocytes migration from the blood stream across the vascular endothelium to support macrophage pick up of ox-LDL-C. VCAM-1 is an endothelial molecule that is expressed on endothelial cells during inflammatory diseases, while IL-1β is the first chemokine produced at the onset of inflammatory response to enhance chemotaxis of immune cells [38]. Interleukin 6 (IL-6) was primarily supposed to have interferon like activity and was named as B cell differentiation factor, but further studies showed its role as a multifunctional cytokine that regulates immune responses, the acute-phase response of the liver, hematopoiesis, and inflammation. One of the essential roles of IL-6 is the promotion of inflammatory reactions through the expansion and activation of T cells, differentiation of B cells, and the induction of acute-phase proteins by hepatocytes [39].
The trends obtained in the serum IL-6 concentration in our study is more likely to be proinflammatory, as other inflammatory markers were not overtly increased to connote massive inflammatory response  [40]. C-reactive protein (CRP) is an acute-phase protein that serves as an early marker of inflammation or infection. CRP binds to phosphocholine expressed on the surface of damaged cells, as well as to polysaccharides and peptosaccharides present on bacteria, parasites and fungi [41]. This binding activates the classical complement cascade of the immune system and modulates the activity of phagocytic cells, supporting the role of CRP in the opsonization (i.e. the process by which a pathogen is marked for ingestion and destruction by a phagocyte) of infectious agents and dead or dying cells. When the inflammation or tissue destruction is resolved, CRP levels fall, making it a useful marker for monitoring disease activity. The patterns we reported in the CRP concentrations in the rats administered aspartame Table 3 supported the oxidative cytotoxicity of aspartame and the antioxidative capabilities of coffee Table 3. In addition, the trend across the groups gave further credence to our finding that the metabolites of coffee might not target primarily the liver, but other tissues in the rats, while those of aspartame might be the liver.
The zinc sulfate turbidity (ZST) test is used to estimate the concentration of serum gamma globulin in a process called turbidimetric [39,40]. The reported concentrations of total immunoglobulins (IGT) in this study Table 3 indicated that there was no massive increase in secretions of immunoglobulins, thus, there was no serious cellular damage or infection, neither was there massive destruction/utilization of immunoglobulins, especially in the coffee administered rats. It can also be inferred that the hepatocytes were preserved as depicted in the coffee administered rats. This is logical because gamma-globulin levels are known to rise in infection, liver damage and or, liver disease, which often result in abnormal zinc sulfate turbidity in most chronic cases [41]. However, the increase in IGT in the H-BMI rat administered aspartame supported the oxidative-cytotoxicities of the metabolites of aspartame. The photomicrographs of the sections of the livers in rats administered coffee and aspartame (Plates 1 -8), revealed that the rats with N-BMI had normal hepatocytes and preserved histo-architecture, while those with H-BMI had mild-moderate infiltration by immune cells, blood congestion with mild derangement in the integrity of the hepatocytes and were aggravated by the administration of aspartame. The overall revelations from the photomicrographs of the liver supported the results of the oxidative and inflammatory indices, as well as that of some cytokines that coffee administration was not toxic to the liver and had antioxidative capabilities. In the same vein, aspartame administration revealed cellular cytotoxicities, whose manifestations were aggravated with H-BMI.

Conclusion
The foregoing inferred that coffee had antioxidative capabilities that mitigated the oxidative cytotoxicity of aspartame and subjects with high body mass index had more risk consuming aspartame as an alternative sweetener. The long-term consumption of aspartame was unhealthy even in normal body mass index subjects; thus, frequent consumption of aspartame was not recommended.

Ethical Approval and Consent to Participate
This study was carried out in accordance with ethical laws on animal handling.

Funding
We did not receive any grant for the conduct of the study.