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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