- •Биологическая химия
- •060101 – Лечебное дело
- •Часть II
- •Contents
- •Cholesterol
- •I. Theoretical part
- •1. Lipids metabolism
- •1.1. Classification of lipids
- •2. Glycolipids (basically glycosphingolipids).
- •Galactosyl ceramide
- •1.2. Digestion and absorption of fats
- •Lipolysis of triglycerides in adipose tissue
- •1.3. Fatty acids oxidation
- •2. Transport of fa inside mitochondrion.
- •3 Ketocyl CoA tyolase Palmitic acid preformed rest Palmityl-CoA Acetyl-CoA
- •Oxidation of unsaturated fatty acids
- •Violations of fatty acids oxidation
- •Acetyl-CoA metabolism
- •1.4. Lipogenesis
- •Regulation of fatty acids synthesis and break down
- •1.5. Metabolism of phospholipids
- •1.6. Essencial fatty acids. Eicosanoids
- •1.7. Cholesterol metabolism
- •Distribution and functions of cholesterol
- •Cholesterol biosynthesis
- •1. Conversion of active acetate into mevalonic acid.
- •2. Squalene formation from mevalonic acid.
- •Regulation of cholesterol synthesis
- •Cholesterol esters metabolism
- •Synthesis of bile acids
- •1.8. Regulation of lipid metabolism
- •1.9. Violations of lipid metabolism
- •Violation of fats adsorption processes:
- •Test questions
- •2. Proteins metabolism
- •2.1. Pathways of proteins degradation
- •2. Digestion of proteins.
- •The selectivity of peptidases
- •2.2. Amino acids metabolism
- •The reactions of amino groups
- •Intramolecular deamination.
- •Reactions of the carboxyl group
- •2. Aminoacyladenylate formation.
- •2.3. Ammonia neutralization in the body
- •Storage and transport of ammonia
- •Fumarate pyruvate aspartate
- •2.4. Violations of nitrogen metabolism
- •2.5. Metabolism of individual amino acids
- •2. Metabolism of sulfur amino acids.
- •3. Metabolism of branched chain amino acids.
- •Leu, Ile, Val α-keto acids acyl-CoA derivatives
- •4. Metabolism of dicarboxylic amino acids
- •5. Metabolism of diaminomonocarboxylic acids.
- •6. Metabolism of phenylalanine and tyrosine.
- •7. Metabolism of tryptophan.
- •2.6. Metabolism of conjugated proteins. Chromoproteins metabolism
- •The degradation of hemoglobin in the tissues (the formation of bile pigments)
- •The hemoglobin biosynthesis
- •2.7. Nucleoproteins metabolism
- •Adenine hypoxanthine; guanine xanthine
- •The synthesis of pyrimidine nucleotides u, c, t
- •I nosine-5'-phosphate Xantosine-5'-phosphate
- •The synthesis of deoxyribonucleotides
- •Test questions
- •3. Template biosynthesis
- •3.1. Biosynthesis of nucleic acids
- •3.1.1. Dna biosynthesis (replication)
- •Synthesis of dna in the rna template
- •3.1.2. Rna biosynthesis
- •Rna synthesis in rna template
- •3.2. Protein biosynthesis
- •The properties of the genetic code
- •Preparatory stage of protein synthesis
- •3.2.1. Translation
- •1. Initiation.
- •2. Elongation.
- •3.2.2. Post-translational modification of proteins
- •Transport of synthesized proteins through membranes
- •3.2.3. Regulation of protein biosynthesis
- •3.3. Genetic engineering
- •Test questions
- •4. Hormones, nomenclature, classification
- •Test questions
- •5. Metabolic processes interaction
- •In the organism
- •Protein and carbohydrate metabolisms interconnection
- •Protein and lipid metabolisms interconnection
- •Carbohydrate and lipid metabolisms interconnection
- •Levels of homeostasis regulation
- •Changes in metabolism during starvation
- •Test questions
- •6. Mineral and water and salt metabolism
- •6.1. Water in the human body
- •6.2. Salt metabolism
- •7. Renal biochemistry. The role of the kidneys in the regulation of water and salt metabolism
- •7.1. Excretory function of the kidneys
- •7.2. Homeostatic function of the kidneys
- •7.3. Metabolic functions of the kidneys
- •7.4. Regulation of water and salt metabolism and uropoiesis
- •Test Questions
- •8. Biochemistry of nervous tissue
- •8.1. Features of metabolism of nervous tissue
- •8.2. Mechanism of nerve impulses conduction
- •Cholinergic synapses
- •Test Questions
- •9. Biochemistry of muscle tissue
- •9.1. Chemical composition of muscle tissue
- •Sources of energy for muscle work
- •9.2. Mechanism of muscle contraction and its regulation
- •9.3. Biochemical changes in muscles within pathology
- •Test Questions
- •10. Biochemistry of extracellular matrix
- •10.1. Structure of extracellular matrix
- •Collagens.
- •Fibril-forming collagens
- •Collagen, forming a tela
- •10.2. Features of metabolism of extracellular matrix Catabolism of extracellular matrix proteins
- •Reparation of damage of the extracellular matrix in norm
- •Biochemical changes of connective tissue with aging
- •The lesions of connective tissue
- •11. Blood biochemistry
- •11.1. Respiratory function of blood. Buffer systems of blood
- •11.2. Blood coagulation system. Changes in pathology
- •Extrinsic and intrinsic pathways of blood clotting
- •Test Questions
- •12. Liver biochemistry
- •12.1. Main functions of the liver
- •The role of the liver in the metabolism of carbohydrates
- •The role of liver in lipid metabolism
- •The role of the liver in the metabolism of proteins and amino acids
- •12.2. Choleresis. Pigment metabolism. Types of jaundice
- •The cleavage of hemoglobin: hemoglobinverdoglobinbiliverdinbilirubin.
- •12.3. Detoxifying liver function
- •Test Questions
- •13. Regulation of calcium and phosphorus metabolism
- •14. Bone biochemistry
- •Test Questions
- •II. Laboratory workshop Laboratory work 1. Lipid metabolism
- •Hydrolysis of milk fat by lipase
- •Test Questions
- •Laboratory work 2. Phospholipids. Cholesterol
- •4.1. Schiff reaction.
- •4.2. Salkowski reaction.
- •4.3. Lieberman-Burchard reaction.
- •Preparation of solutions of cholesterol for the calibration curve
- •Test Questions
- •Laboratory work 3. Digestion of proteins. Determination of acids of gastric contents
- •Experiment 2. Qualitative determination of free hydrochloric acid in gastric juice using indicator congo red.
- •Results of the determination of gastric acidity
- •Test Questions
- •Laboratory work 4. The end products of nitrogen metabolism
- •Test Questions
- •Laboratory work 5. Hormones
- •2.1. Biuretic reaction.
- •2.2. Millon’s reaction.
- •2.4. Geller’s test.
- •3. Qualitative reaction to thyroxine.
- •4. Qualitative reactions to the 11-dehydro-17-oxykortikosteron (cortisone).
- •4.1. The reaction with phenylhydrazine sulfate.
- •4.2. The reaction with Fehling's reagent.
- •Test Questions
- •Laboratory work 6. Mineral and water and salt metabolism
- •1.1. Determination the pH of saliva.
- •1.2. Detection of phosphates in saliva.
- •2.1. Qualitative detection of chlorides in the urine.
- •2.2. Detection of calcium in the urine.
- •2.3. Detection of phosphates in the urine.
- •The composition of mixtures for the calibration curve
- •Test Questions
- •Laboratory work 7. Urine biochemistry
- •3.1. Qualitative detection and quantitative determination of protein in the urine.
- •3.1.1. The test by boiling in weak acid environment.
- •3.1.2. The test by boiling in an acid medium in the presence of saturated sodium chloride solution.
- •3.1.3. Geller’s test.
- •3.1.4. The test with sulfosalicylic acid.
- •3.1.5. Quantitative determination of protein in the urine by the method of dilution (Brandberg - Roberts - Stolnikov method).
- •Determination of protein in the urine using dilution method
- •3.2. Semi-quantitative method for determining glucose and ketone bodies in urine using test strips.
- •3.3. Detection of blood pigments in the urine by boiling with alkali (Geller’s test).
- •Test Questions
- •Laboratory 8. Blood biochemistry
- •1. Buffer properties of blood serum.
- •2. Quantitative determination of total protein in serum according to biuretic reaction.
- •The composition of mixtures for the ployying of the calibration curve
- •3. Determination of calcium in serum by the method of de Waard.
- •Test Questions
- •Laboratory work 9. Detection of bile pigments in urine
- •Test Questions
- •Laboratory work 10. Biochemistry of bone and connective tissue
- •1. Preparation of extracts of bone and teeth tissues.
- •Test Questions
- •Bibliography
- •Biochemistry
- •In Two Parts Part II
- •392008, Г. Тамбов, ул. Советская, 190г
Preparation of solutions of cholesterol for the calibration curve
№ of tubes |
Cholesterol standard solution, ml |
Conc. acetic acid, ml |
Ferric chloride solution, ml |
Cholesterol content in the sample, mg% |
1 |
0,1 |
3,0 |
2,0 |
100 |
2 |
0,2 |
3,0 |
2,0 |
200 |
3 |
0,3 |
3,0 |
2,0 |
300 |
4 |
0,4 |
3,0 |
2,0 |
400 |
5 |
0,5 |
3,0 |
2,0 |
500 |
In 20 minutes mesure the optical dencity of the samples against the control. Build a calibration graph according to the results.
Use the graph to determine the cholesterol content in blood serum. The norm of cholesterol in blood serum is 120-250 mg (3,12-6,50 mmol/l).
Test Questions
1. Why hypercholesterolemia is more common than hypoholesterolemiya?
2. What change will the cholesterol synthesis undergo on a vegetable food diet? Why?
3. What organs synthesize cholesterol for export?
4. How is the main part of cholesterol excreted from the organism?
5. What is the difference between the functions of phospholipids and triacylglycerols?
Laboratory work 3. Digestion of proteins. Determination of acids of gastric contents
Experiment 1. The digestion of proteins by pepsin.
The digestion of proteins begins in the stomach under the influence of gastric juice. Pepsin is an endopeptidase. As a result of protein hydrolysis by pepsin peptones are formed – a mixture of more or less complex polypeptides, as well as a small amount of free amino acids.
2 ml of milk-acetate mixture is poured in every of 4 test tubes. The 1st test tube is placed in a boiling water bath, the 2nd – in a water bath at 400C, the 3rd one – left at room temperature, and the 4th test tube is placed in ice. After 10 minutes, add 0,1 ml of gastric juice in all test tubes and mix. Under the influence of the proteolytic enzyme pepsin soluble milk kazinogen is converted into insoluble pepsin. Fix the time of the appearing of first clots of casein on the walls of the test tubes. Conclude about the optimum temperature of enzyme action.
Experiment 2. Qualitative determination of free hydrochloric acid in gastric juice using indicator congo red.
Normal gastric juice has a strongly acidic reaction (pH about 1) because of the presence of hydrochloric acid (0,4-0,5% HC1). Typically, for analysis gastric contents is used, extracted by the probe after a certain time after the test breakfast. It normally has a pH of 1,5-2,5 and the concentration of HC1 is 0,1-0,2%. In a clinical analysis of gastric juice the following types of acidity are distinguished:
1) "free hydrochloric acid", which is in the free state and dissociates into ions;
2) "bound hydrochloric acid" in the form of salt-like compounds with proteins and products of their digestion;
3) "total hydrochloric acid" which is a combination of free and bound hydrochloric acids;
4) "total acidity", due to the amount of acidic substances (bound and free hydrochloric acid, hydrogen phosphates, and in pathologies lactic acid and volatile fatty acids).
The total acidity of gastric juice in man after a test breakfast is 40-60 conventional units; free acidity is 20-40 units. In some dysfunctions of the stomach, the hydrochloric-acid content of the gastric juice may increase or decrease to the point of complete absence (so-called achylia).
In the acidic environment congo red has a blue color, in slightly acidic, neutral and alkaline media red color. The transition pH range of congo red is between pH 3.0 and 5.2. Such a pH value in gastric juice results in only the free hydrochloric acid.
Pour 1 ml of hydrochloric acid in the test tube and add 1-2 drops of indicator congo red. The same experiment is done with a gastric juice samples (samples 1 and 2). Observe the change of indicator’s color, and infer the presence or absence of hydrochloric acid in the studied juices.
Experiment 3. Qualitative detection of free hydrochloric acid in gastric juice with a tropeolin solution.
The transition pH range of tropeolin indicator is between pH 1,3 and 3,0. When pH is above 3,0 the color of indicator is orange-yellow. Only in the presence of mineral acids (when pH is 1,3 and below), color is crimson-red.
In every test tube 1 ml of samples of gastric juice (1 and 2) and hydrochloric acid and 1-2 drops of tropeolin is poured. Compare the color of the indicator in all the samples and make sure that the crimson-red color appeared only in the presence of hydrochloric acid.
Experience 4. Titration of acids of gastric contents.
Acids of gastric contents are titrated with 0,1 n. sodium hydroxide solution. Use a variety of indicators with different transition pH range: phenolphthalein - 8,2-10,0; congo-red - 3,0-5,2; alizarin red - 4,3-6,3 .
5 ml of gastric contents is poured into a flask. Add 10 drops of 1% alcoholic solution of phenolphthalein, 1-2 drops of 1% solution of congo red and titrate with 0,1 n. sodium hydroxide solution to the transition of the original blue to red. The amount of alkali, used for the titration, corresponds to the amount of free hydrochloric acid and identifies by indicator congo red. Not reaching the level of alkali in the burette to the zero, continue titrating until transition of the color in the persistent crimson. The total number of alkali, since the initial level in the burette corresponds to the total acidity and is detected by phenolphthalein indicator. Measure 5 ml of gastric contents in another flask, add 1-2 drops of 1% aqueous solution of alizarin red and titrate to the transition of the original yellow color to the purple one. The amount of alkali used for titration, corresponds to the sum of all the substances that give an acid reaction, except bound hydrochloric acid, and is detected by alizarin red indicator. Results are entered in Table 5.
Table 5