1

BIOCHEM 6L - 4ABC – Group 8

Metabolic Analysis of BALB/c mice (Mus musculus) exposed in High Fat and Protein Diet
Using Human Diagnostics Test Kits
Tinio, A.I.S.1, Tongson, K-L.R., Tumpalan, K.L.G.1, Tuvera, D.R.B.1, Valenzuela, G.H.1, Veras, L.J.C.1, Vibat, A.A.L.1
1
Department of Biochemistry, Faculty of Pharmacy
University of Santo Tomas

ABSTRACT
The metabolism of an organism changes based on the diet they are exposed in. The body adapts itself to the type
of dietary intake the organism is in which causes its metabolic functions to adjust. This experiment aims to
determine the effects of a high fat, high protein diet to the BALB/c mice models. The blood from the modified
dietary intake was tested in various clinical metabolic and diagnostic tests to determine its difference with those
with the normal diet. The experiment was conducted to prepare the modified pellets suitable for a high fat, high
protein diet. Euthanasia via cervical dislocation was performed and collection of blood was done through
intracardiac puncture. The blood collected was used in 18 assays using the test kits provided by Human Diagnostics
Worldwide. The results obtained in the discussion area were solely based on the test kit assays that were
performed on the blood serum. There were no negative results obtained for each test, therefore in this
experiment, no comparison will be made between the normal diet and the modified diet. In conclusion, the sample
exhibited high amounts of electrolyte (K+, Na+ and Cl-) content, protein content, lipid profile content, acid and
alkaline phosphatase activities and creatinine, urea and uric acid content. The sample exhibited normal to low
levels of aminotransferases activities. The results obtained can be inferred that the test subjects may be on the
verge of acquiring a disease with their high levels of protein content, lipid content and enzymatic activities

INTRODUCTION Changes in the diet may lead to a simultaneous

Metabolism of an organism changes based on occurrence of different medical conditions and
the diet they are exposed in. The body adapts itself comorbidities including obesity, hyperglycemia,
to the type of diet the organism is eating which hypertension or dyslipidemia, which are all
causes its metabolic function to adjust. To determine characteristics of the Metabolic Syndrome, MetS [1].
how the diet, high fat and high protein diet, affects This syndrome is caused by genetic or environmental
the metabolism of the animal model, different clinical factors which include the sedentary lifestyle, physical
laboratory tests were used. These tests include the inactivity and eating habits.
electrolyte contents (Sodium, Potassium and The BALB/c albino mice strain were used in the
Chloride), lipid profile determination (Total, LDL and experiment because this strain is most commonly
HDL cholesterol), excretion of wastes (Urea, Uric acid used for immunological research where it is mostly
and creatinine), phosphatase activity (Acid and susceptible to various infections that may be induced
Alkaline Phosphatase) and aminotransferases (GPT to itself or through the environment [2]. It is
ALAT and GOT ASAT). immunodeficient, easy to breed and has minimal

weight variations between males and females,
making it applicable for this experiment which
involves the difference of its weight due to its diet
[3]. In addition, this mice strain can also be used in
hybridoma and monoclonal antibody production for
cancer therapy and immunology research.
This experiment aims to determine the effects
of the high fat and high protein diet to an organism,
in this case, the mice. The blood from the modified
diet exposed mice will be tested in various clinical
metabolic and diagnostic test to determine its
difference with those with the normal diet.
METHODOLOGY
A. Materials
The following materials and reagents were used
in the experiment: powdered skimmed milk, brown
sugar, margarine and multivitamins for the diet of the
BALB/c mice. An EDTA-coated 1.5-ml centrifuge tubes
were used for the blood extraction of the BALB/c
mice by preparing a 0.5 M EDTA that consists of
dissolving 14.60 grams of ethylenediaminetetraacetic
acid (EDTA) in distilled water with 6M sodium
hydroxide (NaOH). Assay kits from Human
Diagnostics Worldwide were also used to measure
the different metabolic activities of the test subjects.
1. Modified mice feed preparation
The mice feed was modified to induce the effect
of a high fat and high protein diet to the mice. For
every 100 grams of mice feed, it contains an
additional 225 grams powdered skimmed milk, 35
grams white cane sugar, 225 grams salted butter and
15 grams of Revicon forte (Multivitamins, Minerals +
Amino Acids).
2
The 500 grams of mice feed were pulverized
and were mixed with the additional contents
multiplied by 5. The feed was kneaded into a dough
consistency and was formed into pellets. The new
pellets made were dried by placing it in an incubator
at 37 degrees Celsius for 24 hours. After drying, the
pellets were stored in a 4 degrees Celsius refrigerator
to prevent the formation of molds.
2. Animal Handling
BALB/c mice (Mus musculus) were used in the
study. The mice were first acclimatized to their
environment in the animal house for one week and
were being fed with regular mice feed pellets and
distilled water. After acclimatization, the mice were
fed with the prepared modified mice feed with every
day for one week.
Blood was extracted from the mice via
intracardiac extraction and was placed in a centrifuge
tube with 20 microliters of 0.5 M EDTA to prevent
coagulation. The blood was placed in a centrifuge to
separate the serum and the serum was extracted
using a micropipette. The serum was transferred into
another centrifuge tube and placed in the
refrigerator for storage.
B. Methods
1. Assays performed
The assays performed on the blood serum of
modified diet induced mice were both colorimetric
and non-colorimetric. Each test was executed using
the manuals from their respective kits from Human
Diagnostics Worldwide. The following colorimetric
tests with kits were used for the determination of the
metabolic activities of Albumin via Albumin-BCG
Method, Total Protein by Biuret Method, Electrolyte
(sodium, potassium and chloride) Content, Direct and

Total Bilirubin by Modified Jendrassik/Grof Method,
Uric Acid Content with Lipid Clearing Factor, Glucose
Content and Triglyceride Content with Liquid Clearing
Factor by GPO-PAP Method, Creatinine for Kinetic
Measurements-Method, Urea Content, HDL
Cholesterol Content with Liquid Clearing Factor by
CHOD-PAP Method, Urea Content, LDL Content,
Alkaline Phosphatase and Acid Phosphatase by
Orthophosphoric-Monoester Phosphohydrolase
Method.
RESULTS AND DISCUSSION
The following results are based on the test kit
assays that were performed on the blood serum
extracted from the test animal. There were no
negative results obtained for each test which is why
there will be no reference to compare whether the
test results are above or below the normal range.
Some tests would have a reference value specifically
for mice based on the previous researches, but other
tests may not have the reference value.
A. Electrolyte content
1. Sodium
Our body uses sodium to control blood pressure
and blood volume. Sodium is needed for the muscles
and nerves to work properly. It is essential for fluid
balance and cellular homeostasis.
Normal sodium levels are usually between 136
and 145 millimoles per liter (mmol/L). Blood sodium
levels below 136 mmol/L may mean low blood
sodium (hyponatremia). Blood sodium levels greater
than 145 mmol/L may mean you have blood sodium
levels that are too high (hypernatremia).
3
Too much water intake, heart failure and/or
kidney failure because of fluid retention can cause
low sodium level in the blood. A low level can also be
caused by loss of sodium in diarrhea, fluid, and vomit,
or by a deficiency of adrenal hormone. On the other
hand, too much intake of salt or not enough intake of
water can cause a high level. High levels of can cause
target organ damage and may have direct effects on
the brain, heart, kidneys and vasculature [4].
In the test kit, sodium was precipitated from the
blood serum using Mg-uranyl acetate, the uranyl ions
remaining in the suspension will form yellow brown
complex with thioglycolic acid which product will
then be read at 405 nm.
Table 1: Tabulated results for Sodium concentration
determination
Blank Sample Sample Derived Sodium
blank Conc.
0.048 0.052 0.671 23362.5
mmol/L or
46725 mval/L
As observed in the computed values, the
derived sodium concentration was 23362.5 mmol/L
which is very high and may lead to hypernatremia.
This may be due to the natural salt content of the
mice feed together with the salt content of the salted
butter leading to high concentrations of sodium in
the test animal.
2. Potassium
Potassium is one of the essential electrolytes
needed for the homeostasis of different biochemical
systems of the body, most notably the regulation of
action potential occurring within muscle cells and
nerve cells. As all the essential nutrients of our body,
regular uptake of potassium should be monitored.
Low concentrations of potassium in the blood would
 4

cause hypokalemia wherein muscle cells, including
cardiac muscle cells, would be most susceptible to
complications such as muscle cramps, bradycardia,
cardiac arrhythmias and acute respiratory failure if
left untreated. High concentrations of potassium, on
the other hand, would result in hyperkalemia, which
would also cause heart problems such as abnormal
heart contractions, ventricular fibrillation and may
also cause the heart to stop, resulting to death [5].
The reaction principle of the kit involves the the
chelation of free potassium ion with sodium
tetraphenylborate (TPB) to form potassium TPB
(Figure 1).
5 is used to finalize the concentration of K+ in units of
mmol/L.
It was determined that the concentration of K+
in the sample was 108.5 mmol/L. According to
Traslavina et al. (2010), the normal range of K+ levels
in mice blood serum is 3.6 – 5.5 mmol/L. Since the
experiment did not provide any negative controls, it
can be concluded that either the sample was not
properly diluted; thus, needs to be further diluted
and calculated with its corresponding dilution factor,
or K+ levels are very high in the sample obtained. If
the latter is taken note of, it would mean that the
specimen is suffering from hyperkalemia [6][7].
3. Chloride
Chloride plays a dominant role in renal sodium
reabsorption which responds to prostaglandin levels.
It is an essential anion needed for functioning of

Figure 1. Reaction principle of potassium kit test many significant aspects of metabolism, such as,

After reading the sample and standard mixtures aiding in maintenance of the body’s acid-base

at 578 nm, the raw absorbances, Table#:, were used balance [8].

to determine the concentration of potassium present The chloride assay kit is used to measure the

within the serum by using the equation below. chloride in blood and urine. The chloride
concentration is determined between the

Table 2: Raw data from potassium test kit competition of Hg2+ and Fe2+ for 2,4,6-Tris(2-

A578 pyridyl)- s-triazine (TPTZ). The preferred Hg-TPTZ

Standard 0.05 complex exhibits no color. If there is chloride,
Sample 1.085
Hg2+forms HgCl2, which precipitates, allowing TPTZ
to complex with Fe2+ [9].
Table 3: Tabulated results for Chloride concentration
Figure 2. Equation for K+ determination determination
In the equation, c is the concentration of Blank Sample Sample Derived
potassium, in units of present within the sample, blank Chloride Conc.
0.118 0.275 1.959 2528.89 mg/dL
ΔAsx is the absorbance of the sample and ΔAstd is or 712.464
the absorbance of the standard. The numerical factor mmol/L
 5

The derived chloride concentration shows a Hence the chelate complex absorbs light at
high amount of 2528.89 mg/dL chloride. High 540 nm which appears purple. Fasting serum glucose
concentrations may be caused by the production of is higher on a high protein diet [12].
prostaglandins in response to an inflammation within Table 4: Tabulated results for Total protein conc.
the test animal. determination
Blank Standard Sample Derived Total
protein Conc.
B. Protein Assays
0.092 0.176 0.311 14.136 g/dL
1. Total Protein Based on the gathered results, the total protein
The human body uses protein to build and is significant with the high protein diet because
repair tissues. It is also necessary in synthesizing 12.136 g/dL concentration is large enough than the
enzymes and hormones. Protein is an important standard.
building block of bones, muscles, cartilage, skin and
blood. The human body burns carbohydrates for fuel 2. Albumin
and having lesser carbohydrates, the body begins to Mammalian albumins are a family of globular
burn fat. Having a high protein, high fat, low carbs and water-soluble proteins, produced in the liver,
diet decreases energy intake. Excess protein is stored and have the highest abundance in human blood
as fat. However, too much protein can cause certain plasma. The albumin test is done to screen for and
diseases/complications, such as pre-existing kidney help diagnose a liver disorder or kidney disease.
problem can worsen [10]. Sometimes, it is done to evaluate nutritional status,
Biuret protein assay was conducted to especially in hospitalized patients.
determine the presence of peptide bonds in protein Levels of albumin are classified as
and to measure the concentration of total protein. hypoalbuminemia (<2.5 mg/dL), mild
The biuret reagent contains NaOH, hydrated copper hypoalbuminemia (2.5-3.5 mg/ dL), normal albumin
(II) sulfate with potassium sodium tartrate [11]. (3.5-4.5 mg/dL), and hyperalbuminemia (>4.5 mg/dL).
The potassium sodium tartrate when added When albumin level is too low, water can leak out of
to chelate it stabilizes the cupric ions. Where the the blood vessels into other parts of the body and
reaction between cupric ions and nitrogen atoms cause swelling. A low level of albumin in the blood
yields to the displacement of peptide hydrogen under can be caused by malnutrition, too much water in the
alkaline conditions. body, liver disease, kidney disease, severe injury and
Tri- or tetra-dentate chelation with peptide slow bleeding over a long period of time [13].
nitrogen produces the characteristic color; purple – Table 5. Tabulated results for albumin conc.
protein is present (high conc.), blue changes to pink – determination
peptides are present (short-chain peptides – conc. of Blank Standard Sample Derived
peptide bond is low). Albumin Conc.
0.18 0.196 1.383 7.248 g/dL

High protein diet would lead to high albumin
levels since it is water-soluble tissue protein.
Molecules are too large to pass through the kidney’s
glomerular membrane; having more of it in the blood
indicates that the integrity of this membrane is
compromised.
3. Bilirubin
Bilirubin, an orange-yellow pigment, is formed
during metabolic breakdown in the liver. The liver
takes the bilirubin from the blood and changes its
chemical make-up so that most of it is passed
through the poop as bile. Senescent red cells are a
major source of hemeproteins. Breakdown of heme
to bilirubin occurs in macrophages of the
reticuloendothelial system. Unconjugated bilirubin is
transported through the blood to the liver. Bilirubin is
taken up via facilitated diffusion by the liver and
conjugated with glucuronic acid. Conjugated bilirubin
is actively secreted into bile and then the intestine. In
the intestine, glucuronic acid is removed by bacteria.
The resulting bilirubin is converted to urobilinogen.
Some of the urobilinogen is reabsorbed from the gut
and enters the portal blood. A portion of this
urobilinogen participated in the enterohepatic
urobilinogen cycle. The remainder of the
urobilinogen is transported by the blood to the
kidney, where it is converted to yellow urobilin and
excreted, giving urine its characteristic color.
Urobilinogen is then oxidized by intestinal bacteria to
the brown stercobilin.
The bilirubin test is performed on a sample of
blood from the patient. It can chemically determine
the total and if needed, the conjugated (direct) and
unconjugated (indirect) levels of bilirubin in the
blood. Unconjugated bilirubin, which travels in the
6
blood to the liver, is the bilirubin created from red
blood cell breakdown. On the other hand, conjugated
bilirubin is the bilirubin once it reaches the liver and
undergoes a chemical change. This type of bilirubin
moves to the intestines before being removed
through the stool. Since only very little of this form of
bilirubin is present in the blood, a slightly high level
of direct bilirubin indicates a problem with the liver
cells.
Bilirubin test is done in order to help find the
cause of health conditions like jaundice, anemia and
liver disease. For adults over 18, normal total
bilirubin can be up to 1.2 milligrams per deciliter
(mg/dl) of blood. For those under 18, the normal
level will be 1 mg/dl. Normal results for conjugated
(direct) bilirubin should be less than 0.3 mg/dl. Men
tend to have slightly higher bilirubin levels than
women. African-Americans tend to have lower
bilirubin levels than people of other races. If your
bilirubin levels are higher than normal, it is either
your red blood cells are breaking down at an unusual
rate or your liver isn’t breaking down waste properly
and clearing the bilirubin from the blood. On the
other hand, lower than normal levels of bilirubin
aren’t a problem [14].
Table 6. Tabulated results for Bilirubin conc.
determination
Blank Sample Sample Derived
blank Bilirubin Conc.
Total 0.041 0.045 0.086 10.004 umol/L
Direct 0.041 0.034 0.075 7.558 umol/L
Indirect bilirubin was gathered by subtracting
total with direct bilirubin which leads to 2.446 umol/L
indirect bilirubin concentration which is relatively
small.
 7

C. Lipid Profile Determination D-glucono-1,5-lactone and hydrogen peroxide.

1. Glucose Another enzyme, glucose dehydrogenase, catalyzes
Blood glucose is the amount of glucose present the hydrolysis of lactone into D-gluconic acid.
in an individual’s blood. It can be influenced by food Peroxidase enzyme is present in order to break down
and liquids that comes in inside one’s body. Testing the hydrogen peroxide product into water and
one’s glucose levels can identify if an individual has a oxygen. The colored compound is given by the
disease or not. High blood glucose levels can result in reaction of the latter product, oxygen and ortho
hyperglycemia or high blood which is a marker for toluidine. The reaction is read at 500 nanometers
diabetes. However low blood glucose levels aren’t which indicates that the colored compound read at
favorable as well. It can indicate that an individual is this wavelength is proportional to the blood glucose
hypoglycemic which can further result to present in the sample [15].
complications and death.
Table 7. Tabulated results for Glucose conc.
determination
Blank Standard Sample Derived
Glucose Conc.
0.09 0.2973 0.4943 166.26 mg/dL
In glucose test, the gathered value was 166.26
Figure 3: Glucose test kit principle
mg/dL or 9.227 mmol/L. Since these mice weren’t on
2. Triglycerides
their fasted state and the test exhibited is only a
Triglyceride is a compound that is consist of an
random blood glucose testing, they are still under the
ester and three fatty acids. It is the main constituent
normal values of glucose concentration. The mean
of body fat in animals.
for the normal female BALB/c mice blood glucose
For the experiment, high fat and high protein
level is about 193 mg/dL. According to a study,
diet was introduced to the test animal. Based on the
glucose tolerance is more dependent on the
computed value using the formula:
macronutrient taken instead of caloric intake [15].
Since these female mice were given two diets
namely high fat and high protein, high fat might give
them a high blood glucose. The results weren’t the
one that is expected, the blood glucose levels were
just around the normal range for balb/c mice. This is
due to the presence of the high protein diet which
can counteract the effects of high fat diet concerning Figure 4. Triglyceride computation
the blood glucose. The numerical value computed was higher than
For the test’s principle, an enzyme such as the reference. It only means that the diet increases
glucose oxidase is used to oxidize beta-d-glucose into the body fat of the test animal.

The reaction principle behind the assay is the
hydrolysis of triglyceride with lipase. Triglycerides is
hydrolyzed by lipase forming glycerol and fatty acids.
Indicator for the reaction is quinonimine which is
formed from hydrogen peroxide, 4-amino-antipyrine
and 4-chlorophenol catalyzed by peroxidase. The
overall reaction for triglyceride is as follows:
Figure 5. Summary of triglyceride test principle
High protein but low-fat diet in some studies
showed an effective way to improve blood pressure
and level of triglycerides [16]. However, in the
experiment high fat and high protein diet were
prepared for the test animal. Thus, giving a higher
triglyceride content.
3. Total Cholesterol
Cholesterol, a fatty substance that is taken up
by individuals is normally distributed around the
body using blood. In total cholesterol count, two
types of lipoproteins are read. First low-density
lipoprotein which is also called bad cholesterol. This
lipoprotein carries cholesterol through blood from
liver to the cells which is further used for the cells’
stability and fluidity. It is also used in the synthesis of
steroid hormones in the body. Second, high density
lipoprotein which is termed as the good cholesterol
functions as a remedy for atherosclerosis. This
lipoprotein is associated in reverse cholesterol
transport. It gets excess cholesterol and brings it back
to the liver. Hyperlipidemia is an indication of high
levels of cholesterol. This condition can lead to
8
diseases associated with the heart. Although this
condition is detrimental, low levels of cholesterol are
also indications of unhealthiness. Hypolipidemia or
low blood cholesterol is associated with the presence
of another disorder such as anemia, cancer,
hyperthyroidism and others. Both lipoproteins are
synthesized in the liver but for the second type it is
also secreted in the small intestine.
Table 8. Tabulated results for Total Cholesterol
concentration determination
Blank Standard Sample
Derived Total
Chol. Conc.
0.047 0.665 1.009 303.45 mg/dL
or 7.84 umol/L
In the determination of cholesterol level, the
amount of cholesterol computed enters the state of
high cholesterol in the blood which amounts into
303.45 mg/dL or 7.84 umol/l. The female mice were
on a high fat and high protein diet which will surely
make their cholesterol levels high and make them
hyperlipidemic. Since, high protein diet contains high
levels of sodium which is associated with risk of heart
disease, it is not impossible to see elevating
cholesterol levels in the test animals [17]. High
protein diet may also include saturated fat which
lowers HDL levels and raises LDL levels. The other
diet, high fat diet is already expected to give the mice
high cholesterol levels. Induction of large amount of
saturated fats in the diet makes it a candidate for the
cause why cholesterol levels are high.
For the test’s principle, the first enzyme to act
upon cholesterol ester is cholesterol esterase. This
enzyme will hydrolyze cholesterol ester into
cholesterol. The formed product will be oxidized by
cholesterol oxidase which will give ketone-cholest-4-
en-3-one and a hydrogen peroxide. The second

product, hydrogen peroxide will be detected and will
react with 4-aminoantipyrine and phenol. Peroxidase
will catalyze this reaction and will give water and the
colored product, quinonimine. The reaction will be
detected under 500 nanometers since the colored
product is active in this wavelength [17].
Figure 6: Principle of total cholesterol determination
2. HDL Cholesterol
High-density lipoprotein (HDL) is a lipoprotein
which has anti-atherogenic property by reversing
cholesterol transport from the peripheral tissues to
liver. In humans, diets high in saturated fat and
cholesterol raise HDL-cholesterol levels. Low HDL-
cholesterol (<40 mg/dl) and high low-density
lipoprotein-cholesterol (>130 mg/dl) is associated
with the development of coronary heart diseases.
The substitution of fatty acids (FA) for carbohydrates
is beneficially associated with HDL metabolism.
Monounsaturated fatty acid intake may not affect
HDL-C [18].
Chylomicrons, VLDL and LDL are precipitated by
the addition of phosphotungstic acid and magnesium
chloride. The supernatant contains the HDL
cholesterol and was assayed with the cholesterol
liquicolor test kit [19].
The cholesterol esterase hydrolyzes cholesterol
esters to free cholesterol and fatty acids. The free
cholesterol is oxidized to cholesten-3-one and
9
hydrogen peroxide by cholesterol oxidase.
Peroxidase then catalyzes the reaction of hydrogen
peroxide with 4-amintoantipyrine and phenol to
produce a colored quinonimine product.
Table 9. Tabulated results for HDL cholesterol
concentration determination
Blank CAL Sample
Derived HDL
Standard Chol. Conc.
0.094 0.201 0.629 14.14mg/dL or
12.11 mmol/L
The gathered HDL was 14.14 mg/dL which is low
compared to the LDL cholesterol concentration. HDL
cholesterol is known as the good cholesterol because
it brings back the stray LDL cholesterol in
bloodstream back to the liver but since there are a
small amount of HDL in the test animal’s body, there
would be a tendency of accumulation of LDL which
can lead to cardiovascular diseases.
4. LDL Cholesterol
High fat diet will normally show high levels of
LDL (Low density lipoprotein) due to the large intake
of fats which will be stored in the body when the
body do not need energy. Fatty acids are delivered
throughout the body and stored in adipose tissues
through the chylomicrons which will then turn into
HDL, IDL, LDL, and VLDL cholesterol, categorized
based on its density. LDL is also known as the bad
cholesterol because it contains most of the
cholesterol your body doesn’t need anymore.
Too much LDL can form plaques within the
blood vessel, increase the blood pressure and
increase the risk of cardiovascular disease.
The LDL cholesterol liquicolor test kit is a direct
enzymatic assay the same as with the total
cholesterol only that it uses cholesterol esterase and
cholesterol oxidase to remove other cholesterol

besides LDL. It combines two steps: removal of
chylomicrons, VLDL and HDL cholesterol by
enzymatic reactions followed by the determination
of concentration of the remaining LDL cholesterol
using specific surfactants to produce cholestenone
and hydrogen peroxide. The hydrogen peroxide
produced will be combined with the chromogen by
peroxidase to yield a quinone dye which is read at
555 nm wavelength.
Table 10. Tabulated results for LDL cholesterol
concentration determination
Blank CAL Sample
Derived LDL Chol.
Standard Conc.
0.053 0.2783 0.1143 37.52 mg/dL or
0.970 mmol/L
The gathered LDL Cholesterol concentration
against the standard calibrated cholesterol (137.9
mg/dL) was 37.52 mg/dL or 0.970 mmol/L which is
considered high for the mice and may indicate
hyperlipidemia considering its high fat diet.
D. Acid & Alkaline Phosphatases
1. Acid Phosphatase
The acid phosphatase is a ubiquitous lysosomal
enzyme which hydrolyses the organic phosphates at
an acidic pH [20]. This enzyme is mostly found in the
spleen, bone, kidney, liver, intestine, prostate gland
and blood sera. Abnormal high levels of acid
phosphatase may indicate prostate cancer for males;
or, either sickle cell disease or bone disease in
general (Paget’s disease).
Acid phosphatase activity was measured by
using 1-naphthyl phosphate as the substrate, forming
1-naphthol. 1-naphthol will form a complexation
reaction with the FRTR-salt which produces a yellow
colored azo dye. 1-naphthol produced is directly
proportional to the activity of the acid phosphatase
10
in U/I unit. The increase in absorbance at 405 nm is
directly proportional to the acid phosphatase activity.
Figure 7. Acid phosphatase principle
Table 11. Tabulated results for Total acid
phosphatase determination
Time and conditions Total Acid Phosphatase
5 mins at Room Temp 62.124 U/I
3 mins at 37c 67.084 U/I
The acid phosphatase gathered from the serum
was high and there was a significant increase on its
activity after incubation at 37c. Most enzymes are
more active at the physiological temperature,
producing an increase on its activity from 62.124 U/I
to 67.084 U/I.
But, based on the mice results compared to the
human normal range, U/I of acid phosphatase activity
beyond 60 is considered high and may indicate
inflammation or cancer within the animal model.
2. Alkaline Phosphatase
Alkaline phosphatase (ALP) test is used in
tandem with other biochemical tests to quantify the
amount of the enzyme in human blood. Elevated
levels of the enzyme may indicate diseases related to
liver and the gallbladder, such as gallstones [21].
The reaction principle in the test is the
hydrolysis of p–nitrophenyl phosphate by the
enzyme. The resulting products are a free phosphate
group and p – nitrophenol (Figure 8)
Figure 8: Mechanism of enzymatic action of ALP
The amount of p – nitrophenol produced from
the reaction is proportional to the activity of the
enzyme; thus, corresponds to its amount within the
 11

sample. P – nitrophenol was then read at 405 nm Urine:

under UV-Vis spectrophotometric analysis. The
absorbances were then inserted in the equation Creatinine conc urine 24 h:
below to determine the amount of enzyme in units of C = mg/dl x ml urine/24 hh x 0.01
  = 4.55 mg/dl x ____
U/I or IU/L [22]. Figure 10. Creatinine computation
The data gathered was used in the formula and
the value of 4.55 mg/dl which is the creatinine
Figure 9. Equations for ALP determination
concentration of urine for 24 hours.
In the equation, U/I is the traditional unit for
The computed values were higher than the
enzymatic activity that occurred in the sample, ΔA is
reference. It only shows that high fat and high
the difference of the absorbances read at different
protein diet elevates the level of creatinine.
periods of time, and t is time. In the experiment, the
2. Urea
group followed procedure 1, so the first equation was
The urea concentration in the body indicates
used. Both the raw data and the computed results
the amount of nitrogenous waste from the final
are shown below.
breakdown of amino acids. In amino acid breakdown,
Table 12. Tabulated data and results for ALP
ammonia is formed and is highly toxic, thus it cannot
determination
be allowed to accumulate in the body. One way to
Time A405 ∆A ∆ A/min U/I
excrete ammonia is to convert it to urea CO(NH2)2 by
(min)
the liver and excrete it through the bloodstream with
0 0.372 - - -
1 0.398 0.026 0.026 82.96 water. The urea from blood will then pass through
2 0.423 0.051 0.0255 87.541 the kidneys for filtration and excretion of wastes such
3 0.439 0.067 0.0221 75.903
as urea.

E. Creatinine, Urea & Uric Acid High protein diet may cause a significant

1. Creatinine increase of urea especially when the body is using the

Creatinine is excreted by the body by the protein from muscles as energy source which is bad

kidneys. It is the waste product of muscle or protein for the body. High fat diet can also lead to

metabolism. The method used id based on Jaffe ketogenesis and form ketone bodies such as acetone

reaction. The creatinine produces orange-red which is the cause of urea breath.

complex with picric acid in alkaline medium. Thus, In the urea test, urea is hydrolyzed in the

having the reaction: Creatinine + Picric Acid → presence of water and urease which will produce

Creatinine-picrate complex. ammonia and carbon dioxide. It is then followed by a

The creatinine concentration was calculated modified Berthelot reaction. The Berthelot reaction is

using the formula: where the ammonia reacts with hypochlorite, phenol

Serum/plasma: and alkali to produce a blue complex - indophenol. In

this case, the test results with the ammonium ions
 12

were to react with hypochlorite and salicylate to rate, it can still proceed into complications or
form a green dye with the maximum absorbance at diseases such as Hodgkin’s disease, sarcoma and
either 546 or 578 nm. The absorbance obtained is various carcinomas [23].
directly proportional to the urea concentration in the In the uric acid test performed on the female
sample. mice, it was discovered that high fat and high protein
To determine the concentration of the urea diet increases their uric acid levels with a value of
present within the blood serum sample raw 6.32 mg/dL or 376.19 umol/l. Since high fat diet is a
absorbance of the sample was divided with the low carbohydrate diet, it encourages ketosis wherein
standard multiplied by the 13.3 factor. this is the metabolic pathway that produces the
Table 13. Tabulated results for Urea conc. source of energy in the test animals.
determination In ketosis, ketone bodies are formed due to
Blank CAL Sample Derived Urea lipolysis. These products inhibit the excretion of uric
Standard Conc.
acid via urine thus will retain in the blood. Another
0.053 0.2783 0.1143 31.68 mmol/L
suspect for high uric acid levels is the high protein
The resulting concentration of urea was 31.68 diet. High protein diet contains food that is of course
mmol/L which could be due to the excess protein rich in protein. These foods are also rich in purine in
produced which is being turned into waste products which uric acid is produced. Therefore, consuming
to reduce ammonia/ nitrogen in the body. high protein diet elevates uric acid levels which is
3. Uric Acid seen on the test animals [24].
Uric acid is a byproduct when purines from food For the test’s principle, Uric acid in the blood is
or diet is broken down. Liver, dried peas, and subjected into uric acid test which uses the enzyme
mackerel are examples of food that have high purine uricase. The said enzyme oxidizes uric acid into
content. Uric acid in the blood or serum uric acid can allantoin, carbon dioxide and hydrogen peroxide.
help in determining uric acid levels in the body. Peroxidase catalyzes the interaction of 2,6-
High levels of uric acid in the blood can be a sign dichlorophenol together with 4-aminophenozone to
of hyperuricemia which can be asymptomatic or form a colored product which is 4N-(3,5-dichloro-1,2-
symptomatic. Asymptomatic hyperuricemia can be benzoquinone-monoimino)-phenazone. The reaction
developed further into symptomatic ones. was read at 520 nanometers which shows that uric
Symptomatic hyperuricemia exhibits gout, acid is proportional to the uric acid present in the
nephrolithiasis and uric acid nephropathy. However, blood sample [25].
it is also possible to obtain low levels of uric acid
which is termed as hyperuricemia. Hyperuricemia is
achieved when there is a decreased production of
uric acid or increased excretion of uric acid. Though
this metabolic disorder doesn’t show a morbidity
 13

Figure 13. Equation for ALAT determination

In the equation, U/I is the traditional unit for
enzymatic activity that occurred in the sample, ΔA is
the difference of the absorbances read at different
periods of time, and t is time. The numerical factor in
the equation varies with the procedure used and the
size of the kit used.
Figure 11. Reactions in uric acid test The results were then tabulated below for
diagnostic analysis.
F. Aminotransferases Table 14. Tabulated results for ALAT determination
1. GPT ALAT A340 ΔA ΔA/min U/I
0.498 - - -
Alanine aminotransferase (ALAT) or glutamate-
0.516 0.018 0.018 20.718
pyruvate transaminase (GPT) test is used for the 0.528 0.03 0.015 17.265
detection of the enzyme, predominantly found in the
The normal range of ALAT concentration in
liver cells and also found in muscle cells. Alanine
female BALB/C mice is 4.4 – 29.72 IU/L (Ref port).
aminotransferase is used as a biomarker for the early
Based on the calculated results, ALAT concentrations
onset of liver diseases such as hepatitis,
in the serum sample are within the normal range for
hemochromatosis, liver cirrhosis, etc. [26].
mice [27].
The principle reaction involves the
2. GOT ASAT
transamination of L – alanine to 2-oxoglutarate, also
Aspartate aminotransferase (ASAT) or aspartate
known as α-ketoglutarate, to form pyruvate and L-
transaminase, also known as Glutamic oxaloacetic
glutamate (Figure #).
transaminase (GOT) is a liver specific enzyme. It is a
pyridoxal phosphate-dependent transaminase
enzyme.

Figure 12: Mechanism of enzymatic action of GPT This test is used to check for liver damage or

Pyruvate is then reduced to lactate by the injury. The level of ASAT in blood is normally low.

enzyme, lactate dehydrogenase (LDH), present within However, when the liver is damaged, it produces

the buffer reagent. NADH is oxidized to NAD+ during more ASAT in the blood. Thus, elevating the level of

the reaction and the latter was read at 340 nm. After ASAT.

reading the sample under UV-Vis spectrophotometric The reaction involved for the test is:

analysis, the absorbances were used to calculate for

concentration of ALAT in U/I or IU/L.

Figure 14. GOT principle

Table 15. Tabulated results for ASAT determination
 14

Time Blank Sample Derived ASAT https://www.labome.com/method/Laboratory-Mice-

A340 activity (U/I)
and-Rats.html
1 min 0.052 64.565
2 min 0.015 0.055 69.8 [4] Farquhar, W. B., Edwards, D. G., Jurkovitz, C. T., &
3 min 0.056 71.545 Weintraub, W. S. (2015). Dietary Sodium and Health.
Journal of the American College of Cardiology,65(10),
Inferred from the data, the values resulted into
1042-1050. doi:10.1016/j.jacc.2014.12.039
increase of aspartate aminotransferase. It supports
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