ABSTRACT
Monosodium Glutamate is a widely used food additive and flavor enhancer that is present in most soups, salads and processed meat and also present in packaged food without appearing on the label. This could result to inadvertent consumption of monosodium glutamate in high concentrations. The present study investigated the effect of monosodium glutamate on liver and testes of adult male wistar rats, by daily oral exposure of different doses. Wistar rats (n=20) of the average weight of 250-280g were randomly assigned to four group, control, group A, B and C in which (n=5) rats are contained in each group. By the end of the stipulated number of days for the exposure, their organs were subjected to histopathological, biochemical hematological and sperm analysis. The results obtained from these examinations showed the deleterious effect of monosodium glutamate on the liver and fertility. Statistical analysis on sperm motility using ANOVA were carried out and revealed significant difference in the mean percentage of motile cells but no significant difference in the mean percentage of slow motile cells and non motile cells. The liver function parameters revealed no significant difference in the ALP, and AST while there is significant difference in ALT. The hormonal parameters revealed no significant difference in the Luteinizing Hormone, and testosterone but significant difference in the follicle stimulating hormone. However, Monosodium glutamate consumption should be minimized, if not completely avoided to curb its deleterious effect to the hepatocytes and male fertility.
TABLE OF CONTENTS
Title Page
Abstract
Table of Contents
List of Tables
List of Figures
CHAPTER ONE
1.0 Introduction
1.1 Background of the Study
1.2 Statement of the Research Problem
1.3 Research Objectives
1.4 Significance of the Study
1.5 Plan of the Study
CHAPTER TWO
2.0 Literature Review
2.1 Wistar Rat
2.2 Reasons for Mice and Rats in Research
2.3 Monosodium Glutamate
2.4 Uses of Monosodium Glutamate
2.5 Glutamic Acid
2.6 Chemistry of Glutamic Acid
2.7 Optical Isomerism
2.8 Function and Uses of Glutamic Acid
2.8.1 Metabolism
2.8.2 Neurotransmitter
2.8.3 Brain Nonsynaptic Glutamatergic Signaling Circuits
2.8.4 GABA Precursor
2.8.5 Flavor Enhancer
2.8.6 Nutrient
2.9 Liver Function Test
2.10 The Most Common Liver Function Tests
2.10.1AlanineTransaminase(ALT) Test
2.10.2AspartateAminotransferase(AST) Test
2.10.3 Alkaline Phosphatase Test
2.11 Testicle
2.12 Development of the Testis
2.13 The Adult Testis and Male Reproductive Tract
2.14 Sperm Analysis
2.14 Hormones
2.15.1 Testosterone
2.15.1.1 Biological Effect of Testosterone
2.15.2 Follicle-Stimulating Hormone (FSH)
2.15.3 Luteinizing Hormone (Interstitial-Cell-Stimulating Hormone)
2.16 Monosodium Glutamate Linked To Weight Gain
CHAPTER THREE
3.0 Materials and Methods
3.1 Chemicals
3.2 Animals
3.3 Experimental Procedure
3.4 Hematological and Biochemical Studies
3.4.1 Liver Function Test of Rats Treated With Methanol Extract Of Fermented Prosopis Africana Seed
3.4.1.1 Determination of Aspartate Aminotransferase (AST)
3.4.1.2 Determination of Alanine Aminotransferase (ALT)
3.4.1.3 Determination of Alkaline Phosphatase (ALP)
3.4.2 Sperm Analysis
3.4.2.1 Semen pH and Sperm Motility
3.4.2.2 Sperm Viability
3.4.2.3 Sperm Count
3.4.2.4 Sperm Head Abnormality Test
3.4.3 Hormonal Assay
3.4.3.1 Testosterone Determination Method for Testosterone Assay: Ichroma Testosterone Method
3.4.3.2 Follicle-Stimulating Hormone Determination
3.4.3.3 Luteinizing Hormone Determination
3.4.4 Total White Blood Count
3.4.5 Tissue Preparation
3.5 Reagents and Apparatus
3.5.1 Reagents
3.5.2 Equipment
3.5.2.1 Components of Ichroma Testosterone
3.5.2.2 Components of Ichroma Luteinizing Hormone
3.6 Reagents
3.6.1 Ajinomoto
3.6.2 Normal Saline
3.6.3 Calculation for Normal Saline Solution
3.6.4 Proceedure for 10 Liters Normal Saline Solution
3.6.5 Chloroform
3.6.6 10% Neutral Buffered Formalin
3.6.7 DPX Mountant
3.6.8 Ethanol
3.6.9 Xylene
3.7 Equipment
3.7.1 Intravenous Cannula
3.7.2 Syringe
3.7.3 Beaker
3.7.4 Light Microscope
3.7.5 Improved Neubauer Counting Chamber
3.7.6 Hematocrit Tube
3.7.7 Ethylenediaminetetraacetic Acid Bottle
3.7.8 Plain Tube
3.7.9 Measuring Cylinder
3.7.10 Weighing Balance
3.7.11 Autoclave
3.7.12 Sample Bottle
3.7.13 Scalpel Blades
3.7.14 Scissors
3.7.15 Thumb Forceps
3.7.16 Lead Pencil
3.7.17 Mettlers Weighing Scale
3.7.18 Tissue Tek II Embedding Machine
3.7.19 Glass Slides
3.7.20 Cover Slip
3.7.21 Motic Compound Light Microscope
3.7.22 Statistical Analysis
CHAPTER FOUR
4.0 Result
4.1 Sperm Assay
4.2 Liver Function Test
4.3 Testicular Hormone
4.4 Histopathological Examination of the Liver
4.5 Histopathological Examination of Testes
CHAPTER FIVE
5.1 Discussion
5.2 Conclusion
References
Appendices
CHAPTER ONE
1.0 INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Monosodium Glutamate occurs naturally in many foods, such as tomatoes and cheeses. People around the world have eaten glutamate-rich foods throughout history. In 1907, Kikunae Ikeda began a research project to identify the substance in kelp (Laminariaceae) that produced a unique taste favoured in soup stocks in Japan. His research was based on the hypothesis that one or more taste substances may exist in Kelp that could not be categorized as bitter, sour, salty, or sweet (the known basic taste at the time). He named this putative fifth basic taste umami. More generally, Ikeda hoped that, if successful, the results of his research might have a commercial application, such as in a seasoning that would contribute to the improvement of human nutrition in Japan. In 1908, he identified the Umami taste component of kelp as L-glutamate. He filed a patent claim for a process to produce a new seasoning consisting mainly of a salt of L-glutamic acid (Ikeda, 1908). Saburousuke Suzuki, a well-known entrepreneur in the chemical and pharmaceutical industry, then began collaboration with Ikeda to produce and commercialize the seasoning. In 1909, this seasoning was named AJI-NO-MOTO and was registered as a trademark. AJI-NO-MOTO was then known and widely used throughout the world.
1.2 STATEMENT OF THE RESEARCH PROBLEM
Monosodium glutamate was originally designated as a Generally Recognized as Safe (“GRAS”) ingredient by the FDA in 1958, along with other commonly used food ingredients like salt and baking powder (Singh, 2005). Specifically the relevant portion of the United States Code of Federal Regulations states, “It is impracticable to list all substances that are generally recognized as safe for their intended use. However, by way of illustration, the Commissioner regards such common food ingredients as salt, pepper, vinegar, baking powder and monosodium glutamate as safe for their intended use” (FDA, 2017). The safety of MSG has been repeatedly reaffirmed by a number of different sources within the scientific community, including the FDA, since that time. In 1987, the Joint Expert Committee on Food Additives of the United Nations Food and Agriculture Organization and the World Health Organization placed MSG in the safest category of food ingredient (Singh et al., 2005). In addition, a report done in 1991 by the European Communities’ Scientific Committee for Foods confirmed this finding, classifying the “acceptable daily intake” of MSG as “not specified,” which is the most favorable categorization for a food ingredient. The Council on Scientific Affairs of the American Medical Association also weighed in on the issue, stating that glutamate has not been shown to pose a “significant health hazard” in any form (Singh et al., 2005). And yet despite the seemingly general scientific consensus that MSG is safe, the food ingredient has nonetheless been subject to overwhelming controversy in the past several decades.
Moreover, the FDA’s position on MSG labeling has remained relatively static for some time, and yet has become a key component in the larger MSG controversy. The FDA requires labeling of all ingredients in processed and packaged foods. Therefore, whenever MSG is added to a food product, it must be listed on the ingredient list under its common name, “monosodium glutamate.” However, when glutamate-containing ingredients, such as Parmesan cheese, soy sauce and hydrolyzed proteins, are included in a food, they are to be listed by their common name (Singh et al., 2005). The FDA, in 1993, proposed adding the phrase “contains glutamate” to certain protein hydrolysates that contain substantial amounts of glutamate, however this initiative was never finalized. For a food ingredient that has received so many safety approvals and for which there is virtually no confirmed scientific evidence of deaths or serious illness, MSG has nevertheless created what can essentially be termed “mass hysteria” in the general population. MSG has been faulted for a whole host of medical conditions, from headaches to cardiac arrhythmia; it has even been blamed for murder (Warren, 1993). One of the most contested issues that arise in the MSG debate is the question of whether to base findings of MSG safety solely on double-blind scientific studies or to take into consideration the anecdotal evidence. A great deal of the outcry against MSG based on potential adverse health effects relies on these personal accounts of MSG intolerance. These types of reports, though not inherently invalid, do raise scientific concerns in that these episodes cannot be directly linked to the ingestion of MSG, and could in fact be attributed to a variety of other factors. A medical dictionary blurb defines Chinese Restaurant Syndrome as follows:
The syndrome refers to a group of symptoms that can occur after eating Chinese food. The symptoms can include headache, sweating, facial pressure or swelling, nausea, numbness or burning around the mouth, chest pains and heart palpitations. Typically, the symptoms are temporary and not life-threatening, said William Geimeirer, a Wilmington-based allergist. The food additive monosodium glutamate, or MSG, which is commonly used as a food preservative, flavor enhancer or meat ten-derizer, has been implicated but never proven to be the cause, according to the National Institutes of Health. The condition was first reported in 1968, the Institute said. Treatment depends on the symptoms. Most people recover on their own (Singh et al., 2005)
The term “CRS” was first coined in 1968 by Dr. Robert Ho Man Kwok to describe the above-noted collection of symptoms he experienced after eating Chinese food. Anecdotal reports of MSG inducing CRS have been repeatedly subject to scientific examination. The vast majority of these studies have been relatively unfavorable, or at best inconclusive, towards these anti MSG claims. A study by two Italian scientists, P.L. Morselli and S. Garatini of the Institute of Pharmacologic Research in Milan, indicated that CRS may ultimately be a result of “autosuggestion.” In a double-blind crossover study, the two scientists examined 17 males and seven females, between the ages of 18 and 34. The two administered 3 gram doses of MSG via 150ml of beef broth and evaluated the participants every 20 minutes for a three hour period. There were two groups of subjects, one group that received broth with MSG and one group that received broth without MSG. An examination of the test results revealed that the group that had received the broth without MSG reported a number of CRS symptoms, including headache, flushing and tightness in the chest, whereas the group that received the actual MSG broth reported no such symptoms. Other researchers have reached similar conclusions with regard to the scientific link between MSG and CRS. Richard Kenney, MD, of George Washington University has done a number of different studies to examine whether there is in fact any scientifically credible evidence indicating a food intolerance to MSG. In one study, Kenney fed 60 subjects a variety of liquids, including orange juice, black coffee, flavored milk, spiced tomato juice and a two percent MSG solution. Kenney’s results indicated that six subjects reacted to coffee, six to spiced tomato juice and only two subjects responded to the MSG, indicating that “MSG was not unique in producing symptoms typical of CRS.” Kenney did a follow-up double-blind study using subjects who claimed that they suffered adverse reactions after ingesting foods with MSG. The test participants drank a “soft drink” solution for four days, on two of which the solution contained 6 grams of MSG. Once again, Kenney’s results proved unfavorable to the anti-MSG camp. Two of the six participants reacted to both of the solutions (with and without MSG), and the other subjects reacted to neither of the solutions. Indeed, there are number of other studies that have produced similar results, failing to produce the adverse reactions that many individuals associate with dietary intake of MSG. One researcher has attempted to explain the existence of these “CRS-like” symptoms even without exposure to MSG, attributing some of these postprandial adverse reactions to high histamine levels in some foods (Chin, 1989). Of course, these studies and their accompanying results are not without critics. One of the most outspoken opponents of MSG, Dr. Adrienne Samuels, has publicly disapproved of many of these studies on grounds that they have been industry-sponsored, “sloppy in . . . design and execution; focus[ing] on areas which were irrelevant to an understanding of the toxic effects of MSG; and . . . even . . . involved in clear-cut scientific fraud.” Specifically, Samuels suggests that some of the placebo studies were inappropriate since the placebos themselves contained glutamate resulting from manufacture. Samuels and her husband, Jack Samuels, who claims to suffer life-threatening symptoms following ingestion of MSG are by far the most vocal of the anti-MSG activists. Their claims seem to center primarily on the fact that these studies are funded by industry and that the FDA has been bought by these very same industry players. However, there is evidence of studies conducted independent of industry that have resulted in the same dubious conclusions regarding the claim that MSG causes CRS; moreover, there is indication that these anti-MSG activists may sometimes attribute industry ties to those who do not hold them.
The FDA has been repeatedly criticized for not proactively addressing the MSG controversy, for not implementing more stringent regulations and more generally for siding with industry executives. Some have even paralleled FDA’s handling of the MSG issue to its management of silicone breast implants on the grounds that, as with implants, the FDA is exhibiting a preference for “erroneous and in some cases deliberately falsified or deceptive industry data.” (Schwartz, 1992)
However, the FDA has defended its handling of the MSG issue on the grounds that it has appropriately engaged in a process of reassessment and evaluation. Dr. Fred Shank, as the director of the FDA’s Center for Food Safety and Applied Nutrition, commented on the MSG controversy, stating, “the public wants a quick fix: Ban it, remove it, or put a warning label on it.” Though FDA has not taken such definitive actions, it does require that when MSG is added to a food, it be included on the ingredient list using its full name, “monosodium glutamate.” Moreover, the FDA considers it misleading for a product to advertise “No MSG” if it includes other forms of free glutamate, given that the average consumer generally associates the term “MSG” with all free glutamate. In addition, the FDA has repeatedly commissioned studies to reaffirm the safety of MSG. The Select Committee on GRAS Substances (“SCOGS”) of the Life Sciences Research Office (“LSRO”) and the Federation of American Societies for Experimental Biology (“FASEB”) reviewed the health aspects of MSG in two independent studies in 1978 and 1980 as part of FDA’s update of GRAS safety assessments. The Committee concluded that MSG was generally safe at ordinary levels of consumption. The 1980 report did indicate that additional research was necessary to determine whether significantly higher levels of glutamate consumption would produce adverse effects. Taking into account the new studies and the development of additional information regarding the physiological effects of glutamic acid that has accumulated since the publication of the SCOGS reports, combined with the ongoing public concern surrounding this food ingredient, the FDA announced in 1992 that it was contracting with FASEB to review the available scientific data on MSG and to prepare a comprehensive evaluation of glutamate safety.
FASEB REPORT
The FDA specified that this scientific review of MSG was to have five primary objectives:
· To determine whether MSG can induce a complex set of symptoms known as Chinese Restaurant Syndrome, or other serious adverse reactions, after oral ingestion of MSG at levels ranging up to or beyond 5 grams per meal;
· To determine whether MSG as used in the American food supply (including as used in hydrolyzed protein products) has the potential to contribute to brain lesions in neonatal or adult nonhuman primates and whether there is any risk to humans from dietary MSG;
· To determine whether hormones are released from the pituitary of nonhuman primates following ingestion of MSG and whether there exists any comparable risk to humans;
· to define the metabolic basis that might underlie these types of adverse reactions; and
· To compile a report on the findings of the review and evaluation.”
The review was to be conducted in two separate phases – the first being an exhaustive review of the existing scientific literature and the second being a comprehensive evaluation of the safety of MSG using the Phase I results as the focus for the Phase II analysis. The FDA put forth 18 detailed questions regarding MSG that FASEB was to focus on in preparing its report. The questions generally dealt with the possible role of MSG in eliciting MSG symptom complex, the possible role of dietary glutamate in causing brain lesions in humans, any underlying conditions that may predispose an individual to adverse effects from MSG, whether levels of consumption or other factors may affect an individual’s response to MSG and the quality of previous scientific data and safety reviews. The FASEB Report deemed the symptoms associated with MSG as “MSG symptom complex,” a term the Expert Panel preferred over the more popularized CRS which the panel felt was “pejorative” and “not reflective of the extent or nature of the symptoms that have been associated with the myriad of potential exposure possibilities.”
The FASEB final report is detailed and complex, over 350 pages long. The general consensus has been that the report reaffirms the safety of MSG for the general population at normally consumed levels, finding no evidence connecting MSG to any serious, long-term medical problems. Specifically, the report stated that though endogenous glutamate metabolism has been linked to certain neurological diseases, such as Alzheimer’s disease or Huntington’s Chorea, there is no evidence indicating that dietary or circulating MSG or glutamate contributes to changes in brain neurochemistry and therefore chronic consumption of MSG cannot be deemed to contribute to or exacerbate any of these glutamate-mediated neurodegenerative diseases. Moreover, while the Expert Panel indicated that some studies have documented the impact of parenterally administered MSG on the hypothalamus of nonhuman primates, the Panel maintained that no studies performed in the prior fifteen years had indicated the ability of orally ingested MSG to produce lesions or damage nerve cells in nonhuman primates.
The report did, however, indicate possible short-term effects following MSG ingestion in two particular subgroups of the general population:
· Otherwise healthy individuals who, within one hour of exposure to a dosage of MSG greater than 3 grams in the absence of food, experience manifestations of the MSG Symptom Complex; and
· Individuals with severe and unstable asthma who may experience MSG Symptom Complex when given MSG in the absence of a meal containing protein and carbohydrate.
With regard to this latter subgroup, the Expert Panel reviewed 11 available reports regarding the link between MSG and asthma, and found that all of the studies were flawed in some capacity or presented insufficient evidence with which to characterize the patient sample. With respect to this “asthma effect,” the FASEB report recommends additional research.
The Expert Panel maintains that reports of adverse reactions to MSG in the scientific and medical literature are case reports as opposed to experimental studies, and the “majority of these reported symptoms are transient and not life-threatening.” The Expert panel did note two exceptions in the case studies that reported cardiac arrhythmia following ingestion of wonton soup. However, in response to these reports, the Panel notes that “the evidence linking these symptoms in these studies with MSG is presumptive, as neither the glutamate content of the individual food or foods consumed nor the blood glutamate levels or any other corroborative evidence was presented.” Moreover, even with these potential subgroups, the Expert Panel maintains that, with the exception of one study, there is no evidence in humans of response when an MSG challenge is given with a mixed meal.
The Expert Panel declined FDA’s request to determine a reasonable classification scheme for the different types of adverse reactions to MSG, declaring that given the limited state of knowledge and the absence of valid epidemiological data, such a scheme would be premature. The Panel recommended “vigorous research and statistical corroboration” before a valid classification scheme could be designed. The Panel did indicate that adverse reactions were more likely to occur when MSG was ingested in capsule or liquid form on an empty stomach or without food. For purposes of determining an appropriate range of doses and methodology to administer during MSG testing, the Expert Panel recommended a double-blind, placebo-controlled test using 0.5g and 3g doses of MSG.
In summary, given that adverse effects were only seen after ingesting 3 grams or more of MSG on an empty stomach, and that the typical serving of glutamate-treated food contains less than 0.5 grams of MSG, the FASEB Report essentially reaffirms the safety of MSG at normal consumption levels for the general population. The Report does however call for further, more extensive research in certain areas of MSG study, in particular the effect of glutamates on asthmatics.
1.3 RESEARCH OBJECTIVES
Monosodium Glutamate (MSG) is one of the world’s most widely used food additives that enhances food taste and increases appetite. Many anecdotal report have suggested Monosodium glutamate to cause diseases known as Chinese restaurant syndrome but still, the Federation of drug administration have marked monosodium glutamate as a safe food. Thus, Monosodium glutamate is a sodium salt of glutamic acid that has been approved to be a safe food and seen as ‘recorgnized as safe’ list of foods despite the contrary anecdotal report by some as causing a disease as earlier mentioned. Meanwhile, there were a large number of documents available about toxic effects of MSG particularly in children, but few observations had been recorded on the changes occurring in liver and testes following MSG administration. Hence, present study is undertaken to see the sub-chronic effects on histology of liver and testes in adult wistar rat after MSG administation.
1.4 SIGNIFICANCE OF THE STUDY
Monosodium glutamate is commonly marketed as a flavor enhancer and is used as a food additive particularly in West African and Asian dishes (Farombi, 2006). Generally, Monosodium glutamate is accepted as a safe food additive that needs no specified average daily intake or an upper limit intake (Samuels, 1999).
However, inadvertent abuse of this food additive may occur because of its savory, meaty taste and abundance, mostly without labeling, in many food ingredients (Egbuonu, 2009).
This study has become important therefore as to venture, delve into the safety of monosodium glutamate when taken in as a food additive especially in the liver and testes
1.5 PLAN OF THE STUDY
Twenty adult male wistar rats are to be used with a weight range of 250-280g. These rats will then be put into groups of four for each five rats. This is done so as to decongest them and to allow them get acclimatized to the new environment. This acclimatization will be done for four week before carrying out the administration of monosodium glutamate doses on them. Before the administration starts, these rats will be grouped according to their close related weight range of five rats for each group in a total of four groups. There will be the control while the rest of the three groups will serve as the treatment groups. The treatment group will be numbered alphabetically; Group A, Group B and Group C. Group A will be administered 8mg/g body weight of monosodium glutamate. Group B will be given 12mg/g body weight of monosodium glutamate. Group C will be administered 16mg/g body weight of monosodium glutamate. The control group will be given food and water in the same amount given for the treatment group. The administration of these male wistar rats will last for twenty eight days.
At the end of the twenty eight days, the rats will be bled for hematological and biochemical study and will eventually be sacrificed and the needed organs which are the liver and the testes will be taken, preserved in neutral buffered formalin 10% solution and prepared for histopathological analysis. The outcome of the analysis will now be given out as the result and interpreted. These results will be discussed and there will be some conclusion.
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