Catedra Biochimie i Biochimie Clinic

CATEDRA BIOCHIMIE I BIOCHIMIE CLINIC

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FACULTATEA STOMATOLOGIE, ANUL II

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Analizat i aprobat la edin a catedrei

din 29.08.2013, proces verbal nr. 1
eful Catedrei Biochimie i Biochimie Clinic

Tagadiuc Olga, conferen iar universitar, d.h.m.

___________________________
THEME 1
Lipids - classification, role.
Digestion, absorption and blood transport of lipids
Experiment 1: Identification of bile acids.
Method's principle: When sucrose is treated with concentrated H2SO4 oxymethylfurfurol is
formed.It react with bile acid to generate a red-violet complex compound
Procedure: Put into a test-tube 2 drops of bile, 2 drops of sucrose and shake the test-tube. Add 5-6
drops of concentrated H2SO4. Wait 2-3 min for the result.
Result: _______________________________________________________________________
Conclusion: ___________________________________________________________________
Initial level of knowledge:
1. Acylglycerols - representatives, structure, physico-chemical properties, biological role.
2. Phosphoglycerides - structure, physico-chemical properties, biological role.

1.
2.
3.
4.
5.
6.

Self-preparing questions:
Biological functions and classification of lipids.
Dietary lipids and their importance. Digestion and absorption of dietary lipids.
Bile acids - classification, structure and functions. Metabolism of bile acids (general notions).
Disorders of lipid's digestion and absorption. Pancreatic, hepatic and intestinal steatorrhea.
Lipids resynthesis in the mucosal cells of the intestine. Transport of lipids. Plasma lipoproteins
types, composition, biological role and metabolism.
Fat-soluble vitamins: A, D, E, K structure, metabolic role, recommended dietary allowances
and sources. Their impact on oral health.

Case studies:
1. Vegetable oils are liquid triglycerides, while animal fats are solid triglycerides. Write the
complete structure of a triglyceride present in oil and another one present in fat and give their
chemical name.
2. Write he reaction catalyzed by pancreatic phospholipase A2, giving names and structures of
substrates and products. Cobra and bee venoms contain phospholipase A2. How does this
enzyme from the venom injure the bitten person?
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3. What causes steatorrhea? What metabolic disorders can occur in persistent steatorrhea? What
clinical manifestations and biochemical changes may be present depending on the cause
steatorrhea?
Self-assessing tests:
1. Why do saturated fats pack more tightly and have a more solid consistency than unsaturated fats?
a. They contain more nitrogen
b. They are heavier
c. Double and triple bonds cause kinks in the fatty acid tails
d. They have no double or triple bonds to put kinks in the fatty acids
2. Cholesterol, which is the major type of sterol in animals, plays a role in:
a. DNA formation
b. Fat digestion
c. Synthesis of testosterone and estrogen
d. Arteriosclerosis
3. Which lipid is most associated with cell membranes?
a. Wax
b. Sterol
c. Phospholipids
d. Triglyceride
4. In_________cells dietary cholesterol is esterified and packaged into __________ for transport to
the ______________.
a. Hepatic, HDL, intestine
b.Mucosal, chylomicrons, intestine
c. Mucosal, chylomicrons, liver
d.Mucosal, HDL, Intestine
5. Which apoprotien is the ligand for the LDL receptor?
a. Apo AI
b. Apo B-48
c. Apo E
d. Apo 100

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THEME 2
Tissular catabolism of lipids
Experiment 1: Total lipid's assay in blood serum with phosphovanillinic reagent
Method's principle: Serum lipids are hydrolyzed with concentrated H2SO4. The final products react
with phosphovanillinic reagent to form a red complex compound.
Procedure:
Test-tubes
Reagents
Experimental
Standard
Control
1.
Serum
0,02 ml
2.
Standard
0,02 ml
3.
Water
0,02 ml
4.
H2SO4
1,5 ml
1,5 ml
1,5 ml
5.
Boil the solutions for 15 min
6.
Cool the solutions
7.
Hydrolysate
0,1 ml
0,1 ml
0,1 ml
8.
Phosphovanilinic reagent
1,5 ml
1,5 ml
1,5 ml
9.
Mix the solutions and incubate 40-50 min at room-temperature
10.
Measure the extinction of the experimental and standard solutions using control
solutions for comparison, 3 mm cuvettes and green filter.
Calculation: C(g/l) = (A/B) 8, where
A - extinction of the experimental solution
B extinction of the standard solution
8 lipids content in standard solution (g/l)
Result: _______________________________________________________________________
Conclusion: ________________________________________________________________
_________________________________________________________________

1.

1.
2.
3.

Initial level of knowledge:

Fatty acids classification, structure of palmitic, stearic, palmitoleic, oleic, linolic, linolenic and
arachidonic acids.
Self-preparing questions:
Metabolism of triglycerides: catabolism (lipolysis) and synthesis substrates, reactions,
enzymes, regulation.
Glycerol oxidation reactions, enzymes, coenzymes, energetic output of the anaerobic and
aerobic pathways.
Oxidation of the saturated fatty acids with an even number of carbon atoms: steps, reactions,
enzymes, regulation, energetic output.

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4. Peculiarities of the oxidation of the saturated fatty acids with an odd number of carbon atoms
and of the unsaturated fatty acids.
5. Biosynthesis of the saturated fatty acids with an even number of carbon atoms: steps, reactions,
enzymes, regulation.
6. Peculiarities of the biosynthesis of the saturated fatty acids with an odd number of carbon atoms
and of the unsaturated fatty acids.
Case studies:
1. Ingestion of high fat foood leads to their storage in adipose tissue. Explain how occurs
resynthesis of the triglycerides in enterocytes, transport of the triglycerides to adipose tissue and
their synthesis in adipocytes.
2. Intense mobilization of triglycerides from adipose tissue occurs under stress and starvation.
What hormones stimulates tissue lipolysis? What are their mechanisms of action? How are
consumed the products of triglycerides hydrolysis?
3. What will be the consequences of a diet high in fats and dificient in carbohydrates on the
oxidation of free fatty acids? Oxidation of which fatty acids with an even or odd number of
carbon atoms, is more advantageous in the absence of sugars in the diet? Please explain your
answer.
Self-assessing tests:
1. Which of the following is an essential fatty acid?
a. Linoleic
b. Oleic
c. Propionic
d. Lauric
2. Which of the following statements is true?
a. Carnitine is required to transport fatty acids in the bloodstream from storage in the adipose
tissues to peripheral tissues where they are oxidized.
b. Fatty acid oxidation begins with the oxidation of the carbon adjacent to the carbonyl carbon.
c. Lipase catalyzes the oxidation of fatty acids to acetyl-SCoA.
d. Low insulin/glucagon ratios promote the degradation of triglycerides by stimulating lipase.
3. Order the following intermediates of beta-oxidation sequentially:
A. CH3(CH2)4CH=CH-CO-SCoA; B. CH3(CH2)4-CH(OH)-CH2-CO-SCoA
C. CH3(CH2)6-CO-SCoA; D. CH3(CH2)4-CO-SCoA;
E. CH3(CH2)4CH2-CH(OH)-CO-SCoA
F. CH3(CH2)4-CO-CH2-CO-SCoA.
a. c
a
f
b
d
b. c
a
e
f
d
c. c
a
b
f
d
d. d
a
b
f
c

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4. How many molecules of ATP are formed during the complete oxidation of one molecule of lauric
acid?
a. 70 ATP
b. 80 ATP
c. 95 ATP
d. 78 ATP
5. Between meals, ______ levels rise and stimulate ______ to degrade triglycerides.
a. glucagon; lipase
b. insulin; lipase
c. insulin, acetyl-SCoA carboxylase
d. glycogen; lipase
THEME 3
Biosynthesis of lipids
Regulation and disorders of lipid metabolism
Experiment 1: Identification of ketone bodies
Method's principle: Acetone and acetoacetate react with nitroprussiat sodium
Procedure: Use only dry test-tubes!
Put in a test-tube 2 drops of urina, 2 drops of 10% NaOH and 2 drops of 10% nitroprusiat sodium.
Shake the test-tube.
Result: _______________________________________________________________________
Conclusion: ________________________________________________________________
Experiment 2: -lipoproteins assay in blood serum
Method's principle: -lipoproteins react with heparin to form a complex compound, that is
sedimentated with CaCl2. The extent of turbidity is proportional to -lipoproteins content.
Procedure: Put in a test-tube 2 ml of 0,27% CaCl2 and 0,2 ml of blood serum. Shake the solution
and measure the extinction of the solution using CaCl2 solution for comparison (5 mm cuvettes, red
filter). Add 0,04 ml of 1% heparin, shake the solution and exactly 4 min later measure again the
extinction.
Calculation: C(CU) = (E2-E1) 100, where E2 the second extinction, E1 the first extinction
Normal values: 3-4,4 CU
Clinical-diagnostic significance: -lipoprotein content increases in atherosclerosis, jaundice,
diabetes mellitus, obesity etc. -lipoprotein content decreases very rarely, for example, in
plasmocytoma.
Result: _______________________________________________________________________
Conclusion: ________________________________________________________________
_________________________________________________________________

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Initial level of knowledge:

1. Cholesterol and cholesterol esters structure, biological role.

1.
2.
3.

4.
5.
6.

1.
2.

3.

Self-preparing questions:
Cholesterol metabolism. Cholesterol biosynthesis: substrates, stages, reactions of the 1st stage,
enzymes, coenzymes and regulation, utilization and elimination.
Ketone body metabolism. Nature of ketone bodies, formation, oxidation (general notions) and
biological role. Ketonemia and ketonuria.
General notions about the metabolism of phosphoglycerides. Biosynthesis of
phosphoglycerides: substrates, de novo and salvage pathways, enzymes and coenzymes,
regulation. Lipotropic compounds, their role
Hormonal regulation of lipid metabolism. Role of insulin, glucagon, glucocorticoids and
catecholamines.
Interrelations between the metabolism of the carbohydrates and lipids.
Lipid metabolism disorders: dislipidemias (hypo- and hyperlipidemias) and tissue lipidoses.
Changes of the lipid metabolism in diabetes mellitus, alcoholism, atherosclerosis and obesity.
Case studies:
After a meal high in carbohydrates, they are converted into lipids and deposited. Specify the
pathway of conversion of carbohydrates into fat and the site of the process.
After consumption of sucrose, the excess of glucose and fructose is converted into fatty acids in
the liver. Fatty acid synthesis requires acetyl-CoA, ATP and NADPH. Specify how are obtained
these compounds from glucose. What is the subsequent fate of the fatty acids?
Clinical and diagnostic value of determination of serum cholesterol. Causes and mechanisms of
hypercholesterolemia. What type of blood lipoproteins carries cholesterol?

Self-assessing tests:
1. Which statement about this compound is FALSE?
a. This compound is a precursor of sex hormones.
b. This compound is involved in giving stability to the
structure of membranes.
c. This compound is synthesized naturally in the body.
d. This compound is relatively insoluble in water.
e. This compound is one of many classes of strength giving
proteins.
2. Which of the following statements is (are) true?
a. HMG~CoA --> mevalonate is the regulatory step in cholesterol biosynthesis
b. Synthesis of cholesterol begins with the condensation of two acetyl~CoA's in the cytosol
c. Glucagon ultimately leads to decreased HMG~CoA reductase activity
d. Cytosolic NADH is a co-substrate for HMG~CoA reductase

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3. HMG~CoA redutase is inhibited by:

a. Cholesterol
b. Glucagon
c. Both
d. Neither
4. What is the difference between ketosis and ketoacidosis?
a. Ketosis is characterized by high blood ketone levels; ketoacidosis is a concomitant
decrease in blood pH.
b. Ketosis is characterized by high blood levels of acetone; ketoacidosis is the concomitant
increase in the blood level of citric acid.
c. Ketosis is characterized by high blood levels of ketone bodies; the acid-catalyzed oxidation
of these ketone bodies into citric acid cycle intermediates is ketoacidosis.
d. Ketosis is a deficiency in the production of the ketone, acetyl-SCoA. Ketoacidosis is the
concomitant increase in the synthesis of citric acid.
5. Sedentary people with elevated triglyceride levels who continue to eat fatty foods are likely to
have high levels of ____________ in their bloodstream.
a. High-density lipoprotein (HDL)
b. Low-density lipoprotein (LDL)
c. Very-low density lipoprotein (VLDL)
d. Chylomicrons

1.

2.

3.

4.
5.

THEME 4
COLLOQUIUM LIPIDS
Classification of lipids (according to their chemical structure, physicochemical properties and
biological role). The biological role of lipids. Classification and structure of fatty acids.
Essential fatty acids. Chemical structure, properties and functions of triglycerides and
phospholipids.
Digestion of lipids in the gastrointestinal tract: lipolytical enzymes and their functions. Bile
acids role in the digestion and absorption of lipids. Absorption of the products of hydrolysis
of lipids. General notions about resynthesis of lipids in the intestinal epithelium and blood
transport of the resynthetised lipids (plasma lipoproteins).
Catabolism of triglyceride in adipose tissue: partial reactions, enzymes, hormonal regulation
of lipolysis (the effect of epinephrine, glucagon, insulin), the biological role of the process.
The fate of the products of hydrolysis of triglycerides.
Glycerol oxidation: partial reactions, enzymes, coenzymes, the energy balance of aerobic and
anaerobic oxidation of glycerol.
-oxidation of fatty acids (fatty acid activation, transfer them from the cytosol to the
mitochondria, mitochondrial step of -oxidation of fatty acids partial reactions, enzymes,
coenzymes, regulation, biological role). Energy balance.

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6.

7.

8.
9.
10.

11.

12.
13.

14.
15.

1.
2.

3.

4.

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Peculiarities of the oxidation of unsaturated fatty acids and saturated fatty acids with an odd
number of atoms of carbon atoms. Oxidation of propionyl-CoA (schematically till succinylCoA).
Biosynthesis of saturated fatty acids: transport of acetyl-CoA from the mitochondria to the
cytosol, the synthesis of malonyl-CoA, sequence of reactions. The biological role of the
process and regulation. Sources of NADPH.
The biosynthesis of fatty acids peculiarities of the biosynthesis of unsaturated fatty acids
and saturated fatty acids with an odd number of atoms of carbon atoms.
Triglycerides
chemical structure, properties and role. Triglyceride biosynthesis: the
localization of the process, reactions, enzymes, regulation, biological role.
Glycerophospholipids (phosphatidyl serines, phosphatidyl ethanolamines, phosphatidyl
cholines): structure, properties and biological role. Biosynthesis of glycerophospholipids
(scheme), enzymes, cofactors. The lipotropic substances.
Cholesterol and cholesterol esthers: structure, properties, and functions. Cholesterol
biosynthesis: the localization process, phases, phase I reactions (untill mevalonic acid),
regulation and biological role.
Ketone bodies: chemical structure, synthesis in the liver and their utilization in extrahepatic
tissues. Ketonemia and ketonuria, causes and mechanisms of their development.
Structure of biological membranes
fluid-mosaic model (Singer and Nicolson), their
functions and properties (selective permeability, self-assembly, mobility of the components).
Chemical structure and physico-chemical properties of the membrane lipids.
Fat-soluble vitamins (A, E, K): general notion about their structure; biological role. Metabolic
disorders caused by the vitamin A, E and K deficiencies.
Vitamin D: structure, synthesis, and biological role. Metabolic disorders caused by the
hypovitaminosis D.
Additional reading:
Naqvi A.Z., Buettner C., Phillips R.S., et al. -3 fatty acids and periodontitis in US adults. J
Am Diet Assoc. 2010;110(11):1669-75 (http://www.ncbi.nlm.nih.gov/ pubmed/21034880).
da Silva P.C., de Almeida Pdel V., Machado M.A., et al. Oral manifestations of celiac
disease. A case report and review of the literature. Med Oral Patol Oral Cir Bucal.
2008;13(9):E559-562 (http://www.medicinaoral.com/pubmed/medoralv 13_i9_p559.pdf).
Mehra P., Wolford L.M. Serum nutrient deficiencies in the patient with complex
temporomandibular joint problems. Proc (Bayl Univ Med Cent). 2008;21(3):243-247.
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446412/pdf/bumc0021-0243.pdf).
McGuire L.C., Cruickshank A.M., Munro P.T. Alcoholic ketoacidosis. Emerg Med
J. 2006;23(6):417-20.
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC 2564331 /pdf/417.pdf)

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5.
6.

7.

8.

9.

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Fatty Acid Metabolism Disorders. (http://www.scdhec.gov/health/mch/

ch/nbs/docs/manual/Fatty%20Acid%20Metabolism%20Disorders.pdf)
Behrend A.M., Harding C.O., Shoemaker J.D., Matern D., Sahn D.J., Elliot D.L., Gillingham
M.B. Substrate oxidation and cardiac performance during exercise in disorders of long chain
fatty acid oxidation. Mol Genet Metab. 2012;105(1):110-5.
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253922/pdf/nihms329808.pdf)
Zhao W., Varghese M., Vempati P., et al. Caprylic triglyceride as a novel therapeutic
approach to effectively improve the performance and attenuate the symptoms due to the motor
neuron loss in ALS disease. PLoS One. 2012;7(11):e49191.
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492315/pdf/pone.0049191.pdf).
Klop B., Elte J.W., Cabezas M.C. Dyslipidemia in obesity: mechanisms and potential targets.
Nutrients. 2013;5(4):1218-40.
(http://www.ncbi.nlm.nih. gov/pmc/articles/PMC3705344/pdf/nutrients-05-01218.pdf
Keizer H.G. The Mevalonate hypothesis: a cholesterol-independent alternative for the
etiology of atherosclerosis. Lipids in Health and Disease 2012;11:149-157
(http://www.lipidworld.com/content/pdf/1476-511X-11-149.pdf)
Berdal A., Papagerakis P., Hotton D., et al. Ameloblasts and odontoblasts, target-cells for
1,25-dihydroxyvitamin D3: a review. Int J Dev Biol.1995;39 (1):257-62
(http://www.ncbi.nlm.nih.gov/pubmed/7626415)

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