KWASU 2020/2021 Harmattan Semester Examinations

BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

KWARA STATE UNIVERSITY, MALETE

FACULTY OF PURE AND APPLIED SCIENCES
SCHOOL OF BASIC MEDICAL SCIENCES
DEPARTMENT OF MEDICAL BIOCHEMISTRY & PHARMACOLOGY

2020/2021 HARMATTAN SEMESTER EXAMINATIONS

BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

(2 Credit Units)

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Special Instruction(s): Attempt Question 1 and any other THREE (3) questions only.
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

1. (a). Read the paragraph below carefully and use it to answer the questions that
follows.
Sweet potato (Ipomoea batatas) is the 6th most important food crop and possesses
anticancer, antidiabetic and anti-inflammatory properties. This study investigated the
effect of sweet potato wine on altered biochemical indices in cholesterol fed rats.
Wister albino rats weighing between 90-120 g were randomized into 8 groups of 10
rats each and fed separately with fresh sweet potato and sweet potato extract. Rats in
Group 1 (control) were fed commercial diet and were given sterile placebo (distil led
water). Group 2 (negative control) rats were fed commercial diet and 2.5 %
cholesterol. Rats in Group 3 (positive control) were fed commercial diet, 2.5 %
cholesterol and standard red wine (400 mL/ 7 0 g). Group 4-7 were the treatment
groups and were treated as follows: TI (2.5 % cholesterol + 400 mL/70 g potato
wine), T2 (2.5 % cholesterol + 800 mL/70 g potato wine), T3 (400 mL/70 g potato
wine) and T4 (800 mL/70 g potato wine). Group 8 (standard control) were fed
commercial diet and standard red wine (400 mg/70 g). The wines and the cholesterol
were daily administered at 9 am and 4 pm, respectively for 5 weeks and the animal
were sacrificed 24 h after the last administration. The sweet potato wine and extract
restores abnormalities in lipid profiles in the cholesterol fed rats, does not have a
significant effect on the serum renal function biomarkers but the liver function
biomarkers were significantly increased. The study suggests that administration of
sweet potato wine would be beneficial in restoring lipid profiles while eliciting liver
damage but may not have significant effect on kidney function.
i. In a scientific research paper, what will be the name of the paragraph
above?
ii. Suggest the best title for the study?
iii. Identify and write out the following in the paragraph:
a. Background of the study
b. Objective of the study
c. Materials and Methods
d. Results
e. Conclusion
(b). State four purpose of research proposal.
(c). Rewrite the reference below correctly.
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

(i). Effects of ketoconazole in hirsute women. Akalin, S. Acta

Endocrinologica. 124(1):19–22. (1991).
(ii). J Mol Biol. 340:1013-1023. Akbas, G.E., Song, J. and Taylor, H.S.
(2004). A HOXA10 estrogen response element (ERE) is differentially
regulated by 17 beta-estradiol and diethylstilbestrol (DES).
(iii). (2015). Al-Hendy, A., Diamond, M.P., El-Sohemy, A. and Halder, S.K.
1(4): 25-100: 572–582. Dihydroxyvitamin D3 regulates expression of sex
steroid receptors in human uterine fibroid cells. J. Clin. Endocrinol. Metab.
(iv). Ambudkar, I.S. 137:1211–1214. Unraveling smooth muscle contraction:
the TRP link. Gastroenterology. (2009).
(25 marks)
a. i. Abstract (1 mark)
ii. Effect of sweet potato wine on altered biochemical indices in cholesterol fed
rats (2 marks)
iii. a. Background of the study:
Sweet potato (Ipomoea batatas) is the 6th most important food crop and
possesses anticancer, antidiabetic and anti-inflammatory properties. (2 marks)
b. Objective of the study
This study investigated the effect of sweet potato wine on altered biochemical
indices in cholesterol fed rats. (2 marks)
c. Materials and Methods
Wister albino rats weighing between 90-120 g were randomized into 8 groups
of 10 rats each and fed separately with fresh sweet potato and sweet potato
extract. Rats in Group 1 (control) were fed commercial diet and were given
sterile placebo (distil led water). Group 2 (negative control) rats were fed
commercial diet and 2.5 % cholesterol. Rats in Group 3 (positive control) were
fed commercial diet, 2.5 % cholesterol and standard red wine (400 mL/ 7 0 g).
Group 4-7 were the treatment groups and were treated as follows: TI (2.5 %
cholesterol + 400 mL/70 g potato wine), T2 (2.5 % cholesterol + 800 mL/70 g
potato wine), T3 (400 mL/70 g potato wine) and T4 (800 mL/70 g potato
wine). Group 8 (standard control) were fed commercial diet and standard red
wine (400 mg/70 g). The wines and the cholesterol were daily administered at
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

9 am and 4 pm, respectively for 5 weeks and the animal were sacrificed 24 h
after the last administration. (2 marks)
d. Results
The sweet potato wine and extract restores abnormalities in lipid profiles in
the cholesterol fed rats, does not have a significant effect on the serum renal
function biomarkers but the liver function biomarkers were significantly
increased. (2 marks)
e. Conclusion
The study suggests that administration of sweet potato wine would be
beneficial in restoring lipid profiles while eliciting liver damage but may not
have significant effect on kidney function. (2 marks)
(b). State four purpose of research proposal.
i To make the reader to understand :-
- What you are going to do
- Rational of the study
- Objectives of the study
- Methodology
- Expected output
ii To help you organize and develop your topic ideas.
iii To Identify an appropriate supervisor.
iv To convince other people of the merit of your idea.
v To obtain support.
vi Being a basis on which to develop your research.
(Any four = 4 marks)
(c). Rewrite the reference below correctly.
(i). Akalin, S. (1991).Effects of ketoconazole in hirsute women. Acta Endocrinologica.
124(1):19–22. (2 marks)
(ii). Akbas, G.E., Song, J. and Taylor, H.S. (2004). A HOXA10 estrogen response
element (ERE) is differentially regulated by 17 beta-estradiol and diethylstilbestrol (DES).
J Mol Biol. 340:1013-1023. (2 marks)
(iii). Al-Hendy, A., Diamond, M.P., El-Sohemy, A. and Halder, S.K. (2015).
Dihydroxyvitamin D3 regulates expression of sex steroid receptors in human uterine
fibroid cells. . J. Clin. Endocrinol. Metab. 1(4): 25-100: 572–582. (2 marks)
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

(iv). Ambudkar, I.S. (2009). Unraveling smooth muscle contraction: the TRP link.
Gastroenterology. 137:1211–1214. (2 marks)

2. (a). Distinguish between the following:

i. Accuracy and Precision
ii. Null hypothesis and Alternative hypothesis
iii. Random error and Systemic error
iv. Cross sectional study and Randomized controlled study
(b). State four (4) goals of research
(c). List three (3) methods of sampling in biomedical research.
(15 marks)
(a). Distinguish between the following:
i. Accuracy and Precision (2 marks)
Accuracy is a measure of how close a measurement comes to the actual or true value
of whatever is measured.
Precision is a measure of how close a series of measurements are to one another,
irrespective of the actual value.
ii. Null hypothesis and Alternative hypothesis (2 marks)
Null hypothesis states that there is no association between the two factors in question
e.g. exposure and disease of interest.
Alternative hypothesis states that there is an association between the two factors in
question e.g. exposure and disease of interest.
iii. Random error and Systemic error (2 marks)
Random error refer to random fluctuations in the measured data.
Systemic error is error due to the instrument being “out of adjustment.”
iv. Cross sectional study and Randomized controlled study (2 marks)
Cross sectional study a type of observational study that analyzes data from a
population, or a representative subset, at a specific point in.
Randomized controlled study a type of scientific experiment that aims to reduce
certain sources of bias when testing the effectiveness of an agent.
(b). State four (4) goals of research (Any four = 4 marks)
i. to discover new facts.
ii. to verify and test important facts.
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

iii. to analyse an event or process or phenomenon to identify the cause-and-effect

relationship.
iv. to develop new scientific tools, concepts, and theories to solve and understand
scientific and non-scientific problems.
v. to find solutions to scientific, non-scientific, and social problems.
vi. to overcome or solve the problems occurring in our everyday life.

(c). List three (3) methods of sampling in biomedical research. (Any three = 3 marks)
 Simple random sampling
 Purposive sampling
 Stratified sampling
 Area sampling

3. (a). Briefly explain the principles, set-up and operation of Thin Layer Chromatography.
(b). In a tabular form, differentiate between the different forms of column
chromatography.
(15 marks)
a. Briefly explain the principles, set-up and operation of Thin Layer Chromatography.
 Thin layer chromatography (TLC) is a method for identifying substances and testing the
purity of compounds. TLC is a useful technique because it is relatively quick and
requires small quantities of material. Separations in TLC involve distributing a mixture
of two or more substances between a stationary phase and a mobile phase.
 The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on
a plate.
 The mobile phase: is a developing liquid which travels up the stationary phase, carrying
the samples with it.
 Components of the samples will separate on the stationary phase according to how much
they adsorb on the stationary phase versus how much they dissolve in the mobile phase.
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

 To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it
is 0.5 to 1 cm deep in the bottom of the jar.
 Close the jar tightly, and let it stand for about 30 minutes so that the atmosphere in the
jar becomes saturated with solvent.
 With a pencil, etch two small notches into the adsorbent about 2 cm from the bottom of
the plate.
 The notches should be on the edges of the plate, and each notch should be the same
distance up from the bottom of the plate.
 The notches must be farther from the bottom of the plate than the depth of the solvent in
the jar.
 Using a drawn-out capillary tube, spot the samples on the plate so that they line up with
the notches you etched. After preparing the development chamber and spotting the
samples, the plates are ready for development.
 Be careful to handle the plates only by their edges, and try to leave the development
chamber uncovered for as little time as possible.
 When the plates are removed from the chamber, quickly trace the solvent front (the
highest solvent level on the plate) with a pencil.
 If the spots can be seen, outline them with a pencil.
 If no spots are obvious, the most common visualization technique is to hold the plate
under a UV lamp.
 Many organic compounds can be seen using this technique, and many commercially
made plates often contain a substance which aids in the visualization of compounds.
(1 mark each for any 8 steps)

b. In a tabular form, differentiate between the different forms of column

chromatography
Mode or type Stationary phase Mobile Mechanism
phase
Adsorption Solid that attracts the Liquid or Solutes move at different
Chromatography solutes gas rates according to the forces
of attraction to the stationary
phase.
Partition Thin film of liquid formed Liquid or Solutes equilibrate between
Chromatography on the surface of a solid gas the 2 phases according to
inert support their partition coefficients
Ion Exchange Solid resin that carries Liquid Solute ions of charge
Chromatography fixed ions & mobile containing opposite to the fixed ions are
couterions of opposite electrolytes attracted to the resin by
charge attached by covalent electrostatic forces & replace
bonds the mobile counterions.
Molecular Porous gel with no Liquid Molecules separate according
Exclusion attractive action on solute to their size:
Chromatography molecules 1. Smaller molecules
enter the pores of the
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

gel, and need a larger

volume of eluent.
2. Larger molecules
pass through the
column at a faster
rate.
Affinity Solid on which specific Liquid or Special kind of solute
Chromatography molecules are immobilized gas molecules interact with
those immobilized on the
stationary phase

(0.5 marks for each of the three column = 0.5*3*5=7.5marks)

4. (a). Once sample has been selected, it is important to ensure that it does not undergo any
significant changes in its properties from the moment of sampling to the time when the
actual analysis is carried out. How can this be achieved?
(b). What is a standard curve, and how can it be employed in biochemical analysis.
(15 marks)
a. Once we have selected our sample we have to ensure that it does not undergo any
significant changes in its properties from the moment of sampling to the time when the
actual analysis is carried out, e.g., enzymatic, chemical, microbial or physical changes.
There are a number of ways these changes can be prevented.
 Enzymatic Inactivation. Many foods contain active enzymes they can cause changes in
the properties of the food prior to analysis, e.g., proteases, cellulases, lipases, etc. If the
action of one of these enzymes alters the characteristics of the compound being analyzed
then it will lead to erroneous data and it should therefore be inactivated or eliminated.
Freezing, drying, heat treatment and chemical preservatives (or a combination) are often
used to control enzyme activity, with the method used depending on the type of food
being analyzed and the purpose of the analysis (3 marks).
 Lipid Protection. Unsaturated lipids may be altered by various oxidation reactions.
Exposure to light, elevated temperatures, oxygen or pro-oxidants can increase the rate at
which these reactions proceed. Consequently, it is usually necessary to store samples that
have high unsaturated lipid contents under nitrogen or some other inert gas, in dark
rooms or covered bottles and in refrigerated temperatures. Providing that they do not
interfere with the analysis antioxidants may be added to retard oxidation (3 marks).
 Microbial Growth and Contamination. Microorganisms are present naturally in many
foods and if they are not controlled they can alter the composition of the sample to be
analyzed. Freezing, drying, heat treatment and chemical preservatives (or a combination)
are often used to control the growth of microbes in foods (3 marks).
 Physical Changes. A number of physical changes may occur in a sample, e.g., water
may be lost due to evaporation or gained due to condensation; fat or ice may melt or
crystallize; structural properties may be disturbed. Physical changes can be minimized by
controlling the temperature of the sample, and the forces that it experiences (3 marks).
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

b. When carrying out certain analytical procedures it is necessary to prepare standard curves
that are used to determine some property of an unknown material. A series of calibration
experiments is carried out using samples with known properties and a standard curve is
plotted from this data. For example, a series of protein solutions with known concentration of
protein could be prepared and their absorbance of electromagnetic radiation at 280 nm could
be measured using a UV-visible spectrophotometer. (2 marks).
For dilute protein solutions there is a linear relationship between absorbance and protein
concentration:

(1 mark for sketch)

5. (a). What are the applications of SDS-PAGE.

(b). List the merits and limitations of SDS-PAGE.
(c). Why is polyacrylamide used as a gel?
(15 marks)
(a). What are the applications of SDS-PAGE.
i. Determine purity of protein samples
ii. Determine molecular weight of protein
iii. Identifying disulfide bonds between protein
iv. Quantifying proteins
v. Blotting applications
(1mark each*5=5marks)
(b). List the merits and limitations of SDS-PAGE
S/No Advantages Disadvantages
1 Migration is Poor band resolution
proportional to the due to high alkaline
molecular weight operating pH
2 Highly sensitive test, Acrylamide gel is
separates 2% potent
difference in mass neurotoxin chemical
3 Require small Gel preparation is
amount of samples difficult and require
longer time
4 Stable chemically Highly cost
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

cross- linked gel

(6 marks for all the advantages and disadvantages)

c. Polyacylamide is used as a gel because:

i. Chemically inert
ii. Electrically neutral
iii. Hydrophillic
iv. Transparent for optical detection
(1 mark each*4=4 marks)

6. (a). In an end of the year party organized by the National Association of Biochemistry
Students, a lot of assorted foods and drinks were made available. Hassanat took few
apples for an appetizer and for a full meal course, she took a plate of fried rice, slices of
plantain, two wraps of moin moin, three boiled eggs, one lap of chicken and two large
pieces of cow meat. Show with the aid of relevant biochemical pathway (only), the central
converging points in the metabolism of the macromolecular components of the above
meals? (8 marks)
(b) Some communities in Rivers state have been continuously exposed to oil spillage and gas
flaring for over four decades. It was suspected that the eco system has been devastated by
pollution arising from the hydrocarbon pollution. Sandra, a research student of
Biochemistry investigated the effect of the spillage on some animals in the living in the
communities. The Table below show the mean concentrations of lipid peroxidation
product, reduced glutathione and catalase activity determined in the liver of experimental
animals drawn from the communities used for the study.
Table 1: Concentration/Activities of biomarkers in native fowls of the communities
Community GSH (nmol mg MDA (nmol Catalase activity
protein-1) mg protein-1) (μg/mg protein) x103
A 74.36±5.31a 16.11±1.48a 15.45±0.06a
B 84.00±2.89a 18.21± 0.44a 10.37±1.20a
C 49.09±5.34b 35.48±1.06b 3.42±1.25b
D 76.08±2.66a 16.10±1.22a 19,80±3.06a
E 25.18±1.89c 53.10±0.55c 2.0±0.60b
From the data generated by Sandra,
i. which community (ies) may have been affected by the pollution? (2marks)
 KWASU 2020/2021 Harmattan Semester Examinations
BCH413 - Advanced Biochemical Methods and Biochemical Reasoning

ii. deduce reasons to show that the pollution could have affected animals in the
communities identified in (ai) above.
(3 marks)
iii. Suggest a title for this study (2 marks)

a.

8 Marks

b.
i. Which community (ies) may have been affected by the pollution?
Community C and E (2marks)
ii. Deduce reasons to show that the pollution could have affected animals in the
communities identified in (ai) above.
There was in MDA level and decrease in GSH concentration and catalase activity.
(3 marks)
iii. Suggest a title for this study
Effect of exposure to asbestos on serum oxidative stress markers in fowls
(2 marks)