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Masaryk University Biochemistry II Exam Questions Reband Ahmed & Khuram Ahmed Biochemistry II - examination GENERAL MEDICINE DENTISTRY General Medicine 4th semester 2009

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Page 1: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

Biochemistry II - examination

GENERAL MEDICINEDENTISTRY

Khuram AhmedReband Ahmed

General Medicine 4th semester 2009

Page 2: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

1. Factors influencing results of laboratory examination (three phases of examination, biological and analytical factors, sample collection and handling of samples, interpretation of results, reference interval and its calculation, critical difference).

Biological factors can influence the results of labority examinations. Body Weight can affect the concentration of some analytes, by changing their distribution volumes. The serum concentration of cholesterol, LDL-cholesterol, triacylglycerols, uric acid, insulin and cortisol positively correlates with obesity. Exercise can effect blood composition values depending on the duration and intensity, and the physical condition of the patient. Exercise causes a reduction of cellular ATP which increases cellular permeability, leading to increases in serum activites of enzymes an metabolites originating from skeletal muscles. Smoking may affects the level of many analytes by nicotine. Smoking increases the concentration of cholesterol and triacylglycerol. Alcohol affects mainly the metabolism of glucose, and it increases liver enzymes in blood. Stress affects production of hormones. Environmental factors include altitude, ambient temperature and geographical localization.

Analytical factors determine the closeness of the measured value to the true value. Precision is the ability of an analytical method to produce the same value for replicate measurements of the same sample, i.e. agreement between two independant test results. Trueness is the closeness of agreement between the average value from a large series of test results and an accepted reference value. Accuracy is closeness between the result of a measurement and an accepted reference value.

Sample collection involves many reccomendations, the patients are not allowed to eat 10-12 hours before blood collection. They have to exclude fat food and alcohol from their diet. Patients can drink ¼ of a litre of water in the morning before the blood collection. Type of blood collected depends on the test ordered, some specimens must be collected in tubes which have anticoagulants. Time of collection is important because concentration of some substances vary throughout the day. Blood collection is usually performed in the morning. Haemolysis can occur if there is rough handling of the sample, use of incorrect sized needle, moisture in the test tube, or centrifugation at high speed. Transport should be carried out with blood samples at 0c, which is the temperature of thawing ice.

Interpretation of results is most frequently carried out by the comparision with the reference interval. Reference values are required from healthy individuals and patients with relavant diseases. Reference interval includes 95% of results of a reference group. 5% of the results are not included (2.5% of the higest values and 2.5% of the lowest values). Critical difference is expressed as statistically significant difference between the two results of a given laboratory test measured in an individual between the giventime interval. The difference reflects the change in clinical state of the patient.

2. The significance of (both functional and non-functional) enzyme assays in blood serum. Isoenzymes - multiple forms of LD and CK.

Enzymes in blood:TYPE EXAMPLE AFTER ORGAN DAMAGE, ACTIVITY WILLPlasmatic co-agulation factors decreaseSecretory amylase, lipase increaseIntracellular ALT increase

Indirect determination involves calculating catalytic concentration (ukat/l), the product of enzyme reaction is determined. It is used for most enzymes such as ALT and AST.

Direct determination involves mass conc (ug/l), enzyme molecules are determined as antigens. It is used for a few enzymes, e.g. PSA.

Isoenzymes are genetically determined differences in the primary structure. They catalyse the same reaction. They may have different subcellular or tissue distribution. They are usually determined by electrophoresis. Elevated blood values are a specific marker of tissue damage.

General Medicine 4th semester 2009

Page 3: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

LD – Lactate DehydrogenaseLactate + NAD(+) > Pyruvate + NADH + H(+)

Is a tetramer (protein with four subunits), with two different chains (H= heart, M=muscle). It has five isoenzymes, with differing composition of chains: (H4) (H3M) (H2M2) (H1M3) (M4).

LDH-1 and LDH-2 are markers of myocardial infarction. Usually LDH-2 is predominant in serum. A LDH-1 level higher than the LDH-2 level suggests myocardial infarction.

LDH-3 is a marker of lung embolia. LDH-4 and LDH-5 mark skeletal muscle diseases.

CK - Creatine KinaseIs a dimer with two chains (M=muscle, H=heart). It has three isoenzymes, CK-MB, CK-BB, and CK-MM. CK-MB is the major isoenzyme in blood. CK-MB is a marker of myocardial infarction.

3. Provision of glucose in different states, the factors increasing susceptibility of glucose (glucagon, adrenaline, cortisol). Glucosuria.

Glucose is the most common monosaccharise, C6H12O6. Its chemical energy is 17kj/g. Its a fuel source for tissues, especially the brain and erythrocytes. The source of glucose in blood is from dietry saccharides, gluconeogenesis, and glycogenolysis.

Feature I II III IV V

Stage description

well-fed

post resorption

early starvation

prolonged starvation

extreme starvation

Time intervala

0-4 h 4-16 h 16-30 h 2-24 d over 24 d

Origin of Glc in blood

food

liver glycogen gluconeogenesis

gluconeogenesis liver glycogen

gluconeogenesis

gluconeogenesis

Utilization of Glc

all tissues

all tissuesb muscle, ad.t. limited

all tissuesb muscle, ad.t. limited

brain, Ercs, kidney

Ercs, kidney, brain - limited

Energy for brain

Glc Glc Glc Glc, ketone bodies

ketone bodies, Glc

Glucagon binds to receptors in liver, it activates adenylate cyclase, which increases cAMP, this activates cAMP dependant protein kinase A which leads to glucogen phosphorylation.

Cortisol increases blood sugar in response to stress. Substrates from proteolysis in muscle are used in gluconeogenesis. It is also an inducer of enzymes in gluconeogenesis.

Adrenaline secretion is a response to acute stress. It is involved with the breakdown of glycogen in the liver and muscles. Also increases glycolysis in muscles.

General Medicine 4th semester 2009

Page 4: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

Glucosuria is when glucose concentration in urine is higher than 0.8mmol/L. Glucosuria is the excretion of glucose into the urine. Ordinarily, urine contains no glucose because the kidneys are able to reclaim all of the filtered glucose back into the bloodstream. Glucosuria is nearly always caused by elevated blood glucose levels, most commonly due to untreated diabetes mellitus.

4. The basic metabolic disorder in diabetes mellitus: the cause of ketoacidosis or of hyperosmolar coma.

Elevated blood glucose is due to lack of insulin => few insulin-dependant Glut-4 transporters => which enables glucose to enter muscle cells or adipose tissue.

Elevated FFA is due to excess glucagon => which leads to increased lipolysis. (FFA in blood are bound to albumin)

Elevated TAG is due to lack of insulin, which means there isn‘t enough Lipoprotein Lipase, LPL (insulin is inducer of it’s synthesis).

KETOACIDOSIS is a state of elevated concentration ketone bodies. It is due to excess of FA from lypolysis, B-oxidation of their carbon chains gives Acetyl CoA. Acetyl CoA is a precursor for synthesis of ketone bodies in the liver.

HYPEROSMOLAR COMA is when extreme hyperglycemia and dehydration are sufficient to cause unconciousness.

Diabetes Mellitus 1: Due to defficiency of insulin caused by autoimmune attack on B-cells of pancreas. Leads to hyperglycemia, ketoacidosis and hypertriglyceridemia.

Diabetes Mellitus 2:This is genetic, and is due to resistance to insulin. It decreases the ability of target cells (liver, muscles, etc) to react to insulin.

5 Lipids in blood plasma and the major classes of lipoproteins (differences in the lipid and apolipoprotein content, in size, in properties and in electrophoretic mobility, the origin in enterocytes and hepatocytes).

Lipids in blood:Cholesterol (free and esterified) 5mmol/lPhospholipids 2.5mmol/lTriacylglycerols 1.5mmol/lFree Faty Acids 0.5mmol/l

Classes of Lipoproteins: (increasing density, decreasing size)Chylomicrons 85% TAGVLDL 50% TAGLDL 50% cholesterolHDL 50% protein

Lipoproteins consist of a polar surface monolayer (phospholipids, free cholesterol, apoprotein) and a non-polar core (triacylglycerol, cholesteryl ester).

LIPORPOTEIN: ORIGIN: TRANSPORT:Chylomicrons Enterocyte Exogenous TAG from GIT --> tissuesVLDL Liver Endogenous TAG from liver -- > tissuesLDL Blood Plasma Cholesteryl ester --> tissuesHDL Liver Free cholesterol --> liver

CM contains predominantly TAG = neutral molecules (without charge)

General Medicine 4th semester 2009

Page 5: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

They do not move in electric field

6 Transformation of chylomicrons and VLDL.

Chylomicrons are produced in enterocytes, via Apo B48. It is secreted into the lymphatic system and joins the blood via the thoracic duct. Chylomicrons carry dietry TAG to peripheral tissues. In plasma chylomicrons recieve Apo E and Apo C11 from HDL. Apo C11 activates LPL (lipoprotein lipase). LPL is attached to capillary surface in adipose, cardiac, and muscle tissue. Triacylglycerol is hydrolysed to FFA and gycerol. Apo C11 is returned to HDL. Chylomicron particles begin to shrink, remnants bind to APO E receptors in the liver where they are degraded in lysozymes.

VLDL is produced in the liver, it transports endogenous TAG from the liver to peripheral tissues.In plasma they take Apo-C11 from HDL. Triacylglycerol is removed by LPL action. VLDL becomes smaller and more dense, it becomes IDL.IDL takes up cholesteryl ester from HDL and becomes LDL by hepatic lipase.

General Medicine 4th semester 2009

Page 6: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

7 Metabolism of high-density lipoproteins.

HDL particles are made in the liver. Nascent HDL are disk shaped (bilayer of phospholipd and proteins). HDL take free cholesterol from cell membranes. Once cholesterol is taken up it is esterified by LCAT (which is made in the liver and activated by Apo A-1). HDL becomes spherical. Spherical HDL is taken up by the liver and cholesteryl esters are degraded.

Cholesterol + Lecithin > Cholesteryl Ester + Lysolecithin

8 The movements of cholesterol and its elimination. The balance of sterols and the bile acids transformation.

Blood cholesterol is 5mmol/l. Its source is from food (fish, eggs, mayonaise) or biosynthesis from Acetyl CoA (in cytoplasm). A small amount of cholesterol is incorporated into the cell membrane. Some is converted into hormones (steroid hormones). Some is converted into bile acids in the liver. Free cholesterol is immediatedly esterified by ACAT (Acetyl CoA Cholesterol Acyl Transferase) to esterified cholesterol. Cholesterol is eliminated in bile/bile salts.

Intracellular cholesterol descreases HMG-CoA reductase (used for cholesterol synthesis), it decreases synthesis of new LDL receptors (to block LDL intake), and it enhances activity of ACAT (to help make storage).

General Medicine 4th semester 2009

Page 7: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

9 The metabolic interrelationships among body organs predominating in a well-fed state (absorptive phase).

After a typical high saccharide meal, glucose leaves the intestine in high concentrations. Hyperglycemia stimulates the pancreas to release insulin, glucagon release is inhibited. Part of the nutrients are oxidized to meet the immediate energy needs, exessive nutrients are stores as glycogen in liver and muscle, and as TAG in adipose tissue.

During hyperglycemia, GLUT-2 transporters facilitate diffusion of glucose in to B-cells. ATP produced by glycolysis closes the ATP-dependant K+ channel, the resulting depolarization opens voltage-gated Ca2+ channels, and increases the intracellular Ca2+. This is followed by exocytosis of granules containing insulin.

Insulin inhibits secretion of glucagon. It supports the entry of glucose into muscle and adipocytes by GLUT-4 transporters. It promotes glycogen synthesis and storage in the liver and muscle. It inhibits glycogen breakdown. It stimulates glycolysis, and intensifies TAG synthesis in the liver.

10 The metabolic interrelationships among body organs predominating after a brief fast (post- absorptive phase) and during prolonged fasting (starvation).

Post-absorptive phase (early starvation):The post-absorptive phase is the time period from the first feeling of hunger, it doesn’t last more than 10-12 hours. Within one hour after a meal, blood glucose concentration declines. Release of glucagon from A-cell begins, and stimulation of insulin discontinues.

Glycogen antagonises the effects of insulin:- stimulates liver glycogenolysis (inhibits glycogenesis)- supports gluconeogenesis from lactate, glycerol and amino acids- activates mobilization of fat stores

has no influence on skeletal muscle metabolism results in maintaining fuel availability in absence of dietry glucose

General Medicine 4th semester 2009

Page 8: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

Gluconeogenesis occurs 90% in the liver, and 10% in the kidneys. It can be from lactate, glycerol or amino acids.

Glycogenolysis = Glycogen > Glucose-1-phosphate > Glucose-6-phosphate > Free glucose

Fatty acids act as a fuel for muscles: FFA > Acetyl CoA > Citric Acid Cycle > CO2 and energy. They come from hydrolysis of TAG by HSL (hormone sensitive lipase). They can be used for ketogenesis in the liver (a fuel for muscles/brain).

Prolonged fastning (startvation):The prolonged fasting phase’s major goal is to spare glucose and to spare proteins. Tissues use less glucose, they use TAG and KB for energy instead. The brain consumes acetoacetate (30-60%) in place of glucose. After a while, KB are not utilized in the muscles, they are saved to be used up in the brain. Sources of proteins are: intestinal epithelium, digestive enzymes, liver enzymes, and skeletal muscle contractile enzymes.

General Medicine 4th semester 2009

Page 9: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

11 Proteins in human nutrition, the biological value of proteins, nitrogen balance and simple methods for assessing the catabolic periods.

Food proteins, tissue protein proteolysis, and synthesis of non essential amino acids > AMINO ACID POOL.The amino acid pool has 3 main uses:1. Synthesis of specialized nitrogenous products2. Synthesis of tissue/plasma proteins3. Deamination and utilization of carbon skeleton

Digestion of proteins:Stomach: PepsinSmall Intestine: Trypsin, Chymotrypsin, Elastase, Carboxypeptidase A/B, aminopeptidase

GASTRIN is secreted by the stomach. SECRETIN is from pancreatic juice. CCK is a product of pancreatic enzymes.

Endogenous protein degradation is by two methods; lysosomal or ubiqitin proteosome. Lysosomal is non-specific, no ATP is required, and is for extracellular and membrane proteins. Ubiquitin proteosome requires ATP, and is for damaged or regulation proteins.

Biological Value: relative amount N used for endogenous protein synthesis from total N absorbed from foodEgg White 100%Whey Protein 100%Milk Cassein 80%Beef 80%Beans 49%Wheat Four 54%Gelatin 25%

Conversion of amino acids after a meal Glutamate and glutamine are metabolic fuel for the enterocyte In the liver, AA are utilized for synthesis of proteins, glucose, and fatty acids Valine, Leucine and Isoleucine are not metabolised in the liver due to lack of aminotransferase; predominate

in blood High content of NH3 in portal blood is removed by the liver by urea synthesis and is excreted

Catabolic Pathway of NitrogenDietry proteins > AA in GITTransamination of AA in cells > GlutamateDehydrogenation deamination of glutamate > NH3

Detoxifying NH3 > urea

Nitrogen balance – the state of protein nutrition can be determined by measuring the dietary intake and output of nitrogenous compounds. N balance = Nin – Nout Three states are distinguished: 1. Nitrogen balance in equilibrium intake = output2. Positive nitrogen balance intake > output (during childhood growth and pregnancy)3. Negative nitrogen balance intake < output (response to trauma or infection or inadequate intake for requirements, there is a net loss of protein.

Growth/Prenancy Positive Effect Metabolic stress Negative Effect Starvation Negative Effect Incomplete food proteins Negative Effect

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Page 10: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

12 The specific functions of the liver in metabolism, proteosynthesis, and in excretion.

Uptake of most nutrients is from the GIT Intensive intermediary metabolism, conversion of nutrients Controlled supply of essential compounds (glucose, ketone bodies, plasma proteins, etc) Ureosynthesis Biotransformation of Xenobiotics Excretion (cholesterol, bilirubin, hydrophobic compounds, some metals)

Metabolism of saccharidesPrimary regulation of blood glucose concentration – via the glucose buffer functionUptake of glucose and storage as glycogenOR initiation of glycogenolysis and gluconeogenesis

Metabolism of lipidsCompletion and secretion of VLDL and HDLKetogenesis produces ketone bodiesSecretion of cholesterol and bile acids into bile (cholesterol elimination)

Metabolism of Nitrogenous compoundsDeamination of amino acids in excess of requirementsProteosynthesis of plasma proteins and blood-clotting factors – zone 1 periportal areaUptake of ammonium for ureosynthesis – zone 1 periportal areaBilirubin capturing, conjugation, and excretion

Detoxification of drugs, toxins, and excretion of some metals. Transformation of hormones – inactivation of steroid hormones, inactivation of insulin.

13 Ammonium transport, the glutamine cycle and the glucose-alanine cycle.

NH3 in portal blood from: protein putrefication in GITdemaination of Gln/Glu in enterocytes

In saliva from: hydrolysis of urea by oral microfloraIn venous blood from: catabolism of AA in tissues

In urine from: hydrolysis of Gln

Glutamine in MuscleProduced by proteolysisA product of ammonia detoxificationCarrier of NH2 group to liver where NH3 is liberated

Glutamine in enterocyteSource of energy for intestinal mucosa (Gln> 2-OG > CAC)Limited usage of glucose and fatty acids as fuel in enterocytes

Glutamine in brainFormation of glutamine is a way of amonia detoxificationSynthesis occurs mainly in astroglial cellsGlutamate decarboxylation gives GABAGLUTAMATE + NH3 > (glutamine synthase / -H2O) > GLUTAMINE

General Medicine 4th semester 2009

Page 11: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

Glutamine in LiverPeriportal Hepatocytes: Source of ammonia for ureosynthesisPerivenous hepatocytes: a form of ammonia detoxification, released into blood to go to enterocytes and kidneys

Glutamine in kidneysIs an energy sourceGlutamine and Glutamate release ammonium ions which makes the pH of urine acidic

Multiple functions of glutamineSynthesis of proteinsMetabolic FuelSource of nitrogen in synthesis of purines, pyrimidines, aminosugarsSource of glutamate for gaba synthesisSource of ammonium ions in urine

14 Degradation of haemoglobin, formation of bile pigments.

Erythrocytes are taken up by the reticuloendothelial cells by phagocytosis. These are cells of the spleen, bone marrow and Kupffer cells in the liver.

Haemoglobin > (haem oxygenase) VERDOGLOBIN > (lose Fe3 and globin > BILIVERDIN > (biliverdin reductase) BILIRUBIN

Conjugated bilirubin is secreted into the bile. As long as bilirubin remains in the conjugated form it cannot be absorbed into the small intestines. In the large intestines, bacterial reductases and B-glucouroniases catalyse the deconjugation and hydrogenation of bilirubin to mesobilirubin and urobilinogen. Urobilinogen is split into dipyrromethene and this condenses into intensively coloured BILIFUSCINS.

General Medicine 4th semester 2009

Page 12: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

Conjugated Bilirubin > (deconjugation/hydrogenation) > mesobilirubin and urobilinogen > dipyrromethens > bilifuscins

15 Metabolism and excretion of bile pigments. The main types of hyperbilirubinaemia

In blood plasma, hydrophilic bilirubin is unconjugated and is transported as a complex with albumin. Unconjugated bilirubin is non-polar. Hepatocytes convert it into a polar form by conjugation with glucouronic acid so that it may be excreted. Glucosyluronate transferase on ER membranes add the glucouronic group to bilirubin. Conjugated bilirubin is polar and water soluble.

Urobilinogens are partly excreted in the urin and partly excreted in the faeces. In air they are oxidised to a dark brown colour.

Major types of hyperbilirubinaemia:Hyperbilirubinaemia > when serum bilirubin is 20-22umol/l/

Icterus (jaundice) > when serum bilirubin is 30-35 umol/l/

Causes of hyperbilirubinaemia:Prehepatic – increased production of bilirubinHepatocellular – due to inflammation or autoimmune diseasePosthepatic – insufficient drainage of intrahepatic or extrahepatic bile ducts

16 Metabolism of iron (absorption, transfer and distribution in the body, functions, iron balance).

Body contains 4-4.5g of Fe.

Daily supply of iron in a mixed diet is about 10-20mg. From this, only 1-2mg are absorbed.There is no natural mechanism of eliminating excess in the body.

Absorption of Iron in duodenum and jejunum:

Ascorbate or fructose promote absorption aswell as Cu2+. Fe2+ is absorbed much easier than Fe3+. Gastroferrin (component of gastric secretion) is a glycoprotein that bings to Fe2+ maintiaing its solubility by

preventing it from oxidising to Fe3+. Insoluble iron salts are formed from Fe3+. Phosphates, ocalate and phylate form insoluble Fe3+ complexes, this disables absorption.

Transferrin:Is a plasma glycoprotein, serum concentration is 2.5-4g/l. Two binding sites for Fe ions. Biosynthesis of transferring is increased during iron deficiency. Iron is taken up by cells through specific receptor-mediated endocytosis.

Ferritin:One molecule can bing a few thousand Fe3+ ions. When it is not carrying iron it is called Apoferritin.It consists of 24 protein subunits.

Hepcidin:Is a hormone produced in the liver which limits accessibility of iron. Biosynthesis is stimulated in iron overload and inflammations. The same two factors stimulate hepcidin that inhibit transferin. It reduces absorption in the duodenum, inhibits Fe transport across placenta, and prevents release of recyclable iron from macrophages.

General Medicine 4th semester 2009

Page 13: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

17 Biochemical tests used for identification of liver injuries (detection of cell damage, cholestasis, reduced proteosynthetic capacity, etc.).

Plasma markers of hepatocytes membrane integrity:Catalytic concentration of intracellular enzymes in blood increasesEnzyme assays of ALT is most sensitive (0.45-0.9ukat/l)

Tests for decrease in liver proteosynthesis:Serum concentration of albumin, transthyretin, transferring and blood co-agulation factors

Tests for excretory function and cholestasis:Serum bilirubin concentration is measuredSerum catalytic concentration of alkaline phosphatesTests for urobilinogen and bilirubin in urine

18 The metabolism of xenobiotics - stage I of their biotransformation (various types of transformation, examples, mixed-function monooxygenases – function of cyt P450).

Xenobiotics are hydrophobic (lipophilic) compounds present in the environment that cannot be used in normal biological processes – they are foreign to the body. Their elimination depends on their transformation to more hydrophilic compounds. They are excreted in milk, urine, bile or sweat.

Stage 1:The polarity is increased by adding a polar group (usually hydroxylation). Reactions usually take place on membranes of ER, or in the cytoplasm. The first stage may convert the xenobiotic into a more biologically active compound.

Types of biotransformationsHydroxylation (aromatic systems)Dehydrogenation (alcohols, aldehydes)Sulfooxidation (dialkyl sulfides (to sulfoxides) Reduction (nitro compounds (to amines))Hydrolysis (esters)

The overall purpose of the biotransformation of xenobiotics is to reduce their nonpolar character as far as possible. The products of transformation are more polar, many of them are soluble in water. Their excretion from the body is thus facilitated.

Monooxygenases:Catalyse reactions of stage 1, they have low substrate specificity. There are two types; those that contain cytochrome p450 or flavin monooxygenases.

Flavin monooxygenases:Important in the biotransformation of drug containing sulphurous or nitrogenous groups on aromatic rings. It produces sulfoxides and nitroxides.

Cytochrome P450 monooxygenases:Major monooxygenases of ER, over 30 isoforms in humans. Haemoproteins, they are the most versatile biocatalysts in

General Medicine 4th semester 2009

Page 14: BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

the body. Highly active in liver, occur in all tissues except RBC and skeletal muscle. They are inducible/inhibited by certain xenobiotics.

19 The metabolism of xenobiotics - stage II (conjugation). Reaction types, reactant activation, products –examples).

Stage 2:Cytoplasmic enzymes catalyze conjugation of the functional groups, introduced in the first phase reactions, with a polar component (glucouronate, sulphate, Glycine, etc). These products are less biologically active.

It renders xenobiotics more water soluble, to enable excretion. Transferases are cytosolic or bound in membranes of ER, and they catalyse conjugation, acetylation or methylation of polar groups added from phase 1. Reactions are endergonic (require energy), and one of the reactants must be activated.

Reaction type Reagent Group in Xenobiotic

Glucournidation UDP-Glucouronate -OH

Sulfation PAPS -OH

Methylation S-AM -phenolic OH

Acetylation Acetyl-CoA -NH2

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Biotransformation of ethanol occurs mainly in the liver.Ethanol is oxidized to acetaldehyde and then to acetic acid.

CH3-CH2OH + NAD+alcohol DHCH3-CH=O + NADH + H+acetaldehyde

– In peroxisomes, catalase can catalyze oxidation of ethanol by hydrogen peroxide:CH3-CH2OH + H2O2 CH3-

CH=O + 2 H2O

– Microsomal ethanol oxidizing system (MEOS, which contains CYP 2E1) is effective preferably at excess alcohol intake (at blood concentrations higher than 0.2 - 0.5 ‰; Km = 10 mmol/l):

CH3-CH2OH + O2 + NADPH + H+ CH3-CH=O + 2 H2O + NADP+

There are three reactions that give acetaldehyde from ethanol.

– Cytosolic NAD+-dependent alcohol dehydrogenase is the most important, it functions even at low concentrations of ethanol (Km = 2 mmol/l, i.e. 0,1 ‰):

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

20 Alcohols and phenols as xenobiotics and their transformation (ethanol and ethylene glycol, salicylates and acetaminophen).

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Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

21 Principles of metabolism control (control of enzyme activity and of protein synthesis, control of transport across membranes, extracellular signals).

Control of enzyme activity is a more rapid type of control than the control of enzyme synthesis. The enzyme activities can be changed effectively in several ways.

- Activation of proenzymes by partial proteolysis of the proenzymeActive enzymes are formed from proenzyme molecules by irreversible splitting of certain parts in their polypeptide chains. This principle of activation is frequent among proteinases because it prevents unwanted breakdown of proteins.

- Allosteric conrol and cooperative effects of enzymes that consist of several identical subunitsRegulatory enzymes are frequently oligomers that consist of several identical subunits. Their saturation curves are usually sigmoid shaped. Allosteric effectors bind non-covalently at a site other than the active site and may either stimulate or inhibit the activity of the enzyme.

- Control arising from regulatory proteins

- Control by reversible covalent modification of enzymes or of their regulatory proteinsPhosphorylation, catalyzed by protein kinases. Acetylation from Acetyl CoA. Carboxylation of glutamyl in residues side chains.

Transport across membranes is regulated. For example, insulin stimulates glycolysis because it promotes the uptake of glucose by muscle and adipose tissue. Binding of insulin to its receptor leads to rapid increase in the number of GLUT4 transporters in the plasma membrane.

Transduction of extracellular signals is important for the cell in receiving and responding to information from the environment. Proteins and small polar signal molecules bind on to specific membrane receptors, which results in a conformational change of the intracellular domain, resulting in the increase of secondary messenger molecule or activation of a protein kinase. Non-polar signal molecules diffuse through plasma membrane and bind to specific proteins called intracellular receptors.

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Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

22 General features of hormone synthesis, secretion, transport, and inactivation in relation to signal intensity received by the target cell.

Hormone synthesis: Protein and peptide hormones are synthesized on the rough ER and in different endocrine cells. They are first “secreted” as large proteins which are biologically inactive – prohormones, which start to get smaller in the ER. Prohormones are transferred to the golgi apparatus for packaging into secretory vesicles. In these vesicles, enzymes cleave the prohormones to produce smaller, biologically active hormones and inactive fragments. >Vesicles are stored within cytoplasm or in the cell membrane until their secretion is needed => exocytosis. Stimulus of exocytosis can be increased by depolarisation of the plasma membrane => Hormone secretion.

Hormone secretion - feedback control of hormone secretion

-ve feedback control - ensure proper level of hormone activity at the level of the target tissue; After a stimulus causes release of the hormone, conditions or products resulting from the action of the hormone tend to suppress its further release – prevents over secretion or over activity.

+ve feedback control – occurs when the biological action of the hormone causes different additional secretion of the hormone; e.g. Luteinizing hormone is secreted as result of the stimulating effect of estrogen from the anterior pituitary before ovulation. LH increases when estrogens increases in the ovaries.

Transport of hormones into blood:

Water-soluble hormones are dissolved in the plasma and transported from their sites of synthesis to target tissues, where they diffuse out of the capillaries, into the intestinal fluid, and eventually to target cells.

Steroid and thyroid hormones circulate bound to plasma proteins.

Inactivation of hormones – there are two main factors increasing or decreasing the concentration of hormones in blood: 1. rate of hormone secretion into the blood and 2. rate of removal of hormone from the blood – metabolic clearance rate. Metabolic clearance rate = rate of disappearance of hormone from plasma (conc. of hormone / ml of plasma). Ways of clearance: => metabolic destruction by the tissues, => binding with the tissues, => excretion by the liver into bile, => excretion by the kidneys into urine

Hormones can be degraded of their target cells by enzymatic processes that cause endocytosis of the cells membrane hormone-reseptor complex the hormone is then metabolized in the cell, and receptors are recycled back to the cell membrane.

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Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

23     Membrane receptors cooperating with G-proteins (types of receptors and G-proteins, corresponding intracellular messengers).

Types of membrane receptors

1. Ion channel receptors mediated by neurotransmitters in synapses – quick responses.2. G-protein linked receptors – “G” because they bind GDP and GTP - result in specific ligand binging in: Stimulate/inhibit phospholipase C Stimulate/inhibit phosphodiesterase Stimulate/inhibit phosphodiesterase3. Receptors with enzyme activity – granylate cyclase4. Receptors activating non-receptor tyrosine kinase activity

G-proteins (response in a few minutes)- GTP/GDP binding proteins- Freely membrane bound (can move along the inner surface)- Participate in various types of second messenger production- All have a similar structure and mechanism of activation- Heterotrimers consist of subunits A, B, and Y

G-protein linked receptorsAll have some common structural features:1) extracellular parts are slightly glycosylated, have accessory binding sites for agonist2) membrane parts: 7 a-helical segments span the membrane, connected by intra and extracellular hydrophilic loops3) intracellular parts, which have the bingind site for a specific G-protein type

G-protein activation- Resting state = a-unit has GDP attached- Hormone binds to extracellular part, makes a complex with the receptor, and GDP is phosphorylated to GTP- The a-GTP interacts with the effector enzyme – activate/inactivated enzyme which causes an increase or decrease in secondary messenger signal

EXAMPLE: receptors with adenylate cyclase system

- membrane bound receptor that catalyses ATP > cAMP + PPi

- cAMP is a secondary messenger

- Gs-protein stimulates adenylate cyclase, so the cAMP increases

- cAMP activates PKA, which is used in phosphorylation reactions

- Gi-protein inhibits AC – opposite effect

Gq-protein stimulates phospolipase C

Gt-protein stimulates cGMP phosphodiesterase

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24      Plasma membrane phosphatidylinositols and the phosphoinositide cascade, the role in signal transduction.

Inositol sources: exogenous (plant food) and endogenous (Glucose-6-phosphate)

Cascade:

Signal molecule binds to the receptor

The receptor activates the G-protein

Activated G-protein (a-unit and GTP) activates the effector = phospolipase C

Phospholipase C catalyses the hydrolysis of PIP2 > DG + IP3

DG and IP3 are secondary messengers

DG activates PK C – phosporylations in the presence of Ca2+

IP3 opens Ca2+ channels in ER > cytosol Ca2+ concentration increases

Ca2+ is associated with calmodulin

Calcium-calmodulin complexes activate calmodulin dependant kinases

Phosphorylated intracellular proteins carry out a biological response to the signal molecule

Enzymes for glycogenolysis and gluconeogenesis are activated by phosphorylation.

Enzymes for glycogen synthesis, glycolysis, FA synthesis and cholesterol synthesis are inactivated by phosphorylation.

Phosphatidylinositol:

Phosphatidate is esterified with myo-inositol

PIP2 is a part of membranes

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25      Protein kinases (main classes) and phosphoprotein phosphatases, regulation of their activity.

Reversible phosphorylation of proteins is intracellular and ATP is the phosphate donor. Phosphorylation is catalysed by highly specific protein kinases. Protein kinases are the largest family of homologous enzymes, there are over 550 human types.

There are two sites where proteins can be phosphorylated:

1. On the serine/threonine residues (alcoholic groups)2. Tyrosine residues (phenolic hydroxyl)

They are both at specific positions in the polypeptide chain.

The signal that activates PK is amplified causing phosphorylation of numerous protein molecules.

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Dephosphorylation of phosphoproteins is carried out by PHOSPHOPROTEIN PHOSPHATASES, and it involves the hydrolysis of the ester bond.

Because protein kinases have profound effects on a cell, their activity is highly regulated. Kinases are turned on or off by phosphorylation (sometimes by the kinase itself - cis-phosphorylation/autophosphorylation), by binding of activator proteins or inhibitor proteins, or small molecules, or by controlling their location in the cell relative to their substrates.

26      Insulin (synthesis, regulation of secretion, fate, insulin receptor and results of its activation). Oral glucose tolerance test.

Synthesis:

In B-cells, islets of langerhans, within the pancreas. Preproinsulin is produced in the endoplasmic reticulum. It is a single peptide. Cleavage of the single peptide and formation of disulphide bonds makes Proinsulin. This passes to the golgi, where it is placed in to vesicles called B-granules. After cleavage of the C-peptide, mature insulin is formed in the B-granules. It has two peptide chains held together by disulphide bridges.

Secretion:

Secreted in response to increase in blood glucose levels. Stimulates glycolysis, lipogenesis, and glycogen synthesis and storage in the liver. Inhibits gluconeogenesis, glycogenolysis and lipolysis.

Degredation:

Insulin binds to receptor (in liver or kidney) and enters the cell by endocytosis of the insulin-receptor complex. Insulase acts on the complex, breaking it down.

Regulation of secretion:

1. Increased blood glucose levels is a signal for increased secretion2. Increased amino acids in plasma after ingestion of proteins also increases secretion3. Gastrointestinal horomone secretin, released after ingestion, causes anticipatory rise

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Examples of regulation by reversible phosphorylation:2 Regulation of enzyme activity

Activated by phosphorylation Inhibited by phosphorylation glycogen phosphorylase-b-kinaseglycogen phosphorylase(glycogenolysis)

glycogen synthase(glycogen synthesis) fructose 2,6-bisphosphatase(gluconeogenesis) fructose 6-phosphate 2-kinase pyruvate dehydrogenase (glycolysis)

acetyl-CoA carboxylase(fatty acid synthesis) HMG-CoA reductase (cholesterol synthesis)

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Receptor:

Transmembrane receptor, activated by insulin Belongs to tyrosine-kinase receptors Insulin binds to receptor Starts many protein activation cascades, translocation of GLUT4 to plasma membrane

oGTT – oral glucose tolerance test:

Used when increased concentration of fasting glucose is found in the serum/plasma. It tests the effectiveness of glucose metabolism.

Procedure:

Blood sample is taken after overnight fasting (10-14 hours) 75g of glucose in 300ml tea Blood sample is taken every 1-2 hours after drinking the tea

Normal values 0 hours 1 hour 2 hoursNormal <6 <11 <8Impaired >6 >11 8-11Diabetes mellitus >7 >11 >11

27      Intracellular hormones receptors, their activation and consequences.

Lipophilic hormones diffuse through the plasma membranes to bind to receptors in the cytoplasm or in the nucleus of target cells. The hormone-receptor comlex under goes activation reaction.

The hormones bound to their transport proteins in the blood, attach to the megalin transport protein and passes into the cytoplasm. In lysosomes the hormone is released from its binding protein via hydrolysis and the hormone binds with its intracellular receptor.

The intracellular hormone-receptor complex binds to DNA sequence HRM (hormone response element) – works as enhancer supporting initiation of transcription on the promoter.

Gene transcription effect and production of target mRNA- Amount of specific protein changed- Metabolic processes are influenced

Low density lipoprotein-related protein 2 also known as LRP2 or megalin is a protein which in humans is encoded by the LRP2 gene.

Function: LRP2 is multiligand binding receptor found in the plasma membrane of many absorptive epithelial cells. LRP2 is a member of a family of receptors with structural similarities to the low density lipoprotein receptor (LDLR). LRP2 functions to mediate endocytosis of ligands leading to degradation in lysosomes or transcytosis. LRP2 (previously called glycoprotein 330) together with RAP (LRPAP1) forms the Heymann nephritis antigenic complex. LRP2 is expressed in epithelial cells of the thyroid (thyrocytes), where it can serve as a receptor for the protein thyroglobulin (Tg).

28 The role of hypothalamic and pituitary hormones – a brief survey, functions.

Hypothalamus – affects the endocrine system, controls emotional behaviour. Most hypothalamic hormones go to pituitary via hypophyseal portal system. It maintains homeostasis, including blood pressure, heart rate and temperature regulation.

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Hypothalamic hormones control the release of the anterior pituitary gland hormones and the hormones of the posterior pituitary gland are synthesized in the magnocellular neurons in the hypothalamus.

The pituitary gland secretes hormones regulating homeostasis, including trophic hormones that stimulate other endocrine glands. It is connected to the hypothalamus by the medial eminence.

Name Location FunctionCorticotropin-releasing hormone paraventricular nuclues with ADH, stimulates anterior pit. To secrete

ACTHDopamine arcuate nucleus inhibits anterior pit. Secreting prolactinGonadotropin-releasing hormone arcuate nucleus stimulates anterior pit. To secrete LH and FSHGrowth hormone releasing hormone arcuate nucleus stimulates anterior pit. To secrete GHVasoprissin (ADH) paraventriculat nuclues with CRH, stimulates anterior pit. To secret

ACTH

ACTH, adrenocorticotropic hormone, polypeptide – secretion of glucocorticoids. Beta Endorphins, polypeptide – inhibits perception of pain.Prolactin, polypeptide – milk production in mammary glands.TSH, thyroid stimulating hormone, glycoprotein – secretion of thyroid hormones.Growth hormone, glycoprotein – promotes growth and lipid/carb metabolism.

29 Synthesis of thyroid hormones (description, localization, secretion and its control).

Thyroxine (T4) – tetraiodothyronine and it’s active form triiodothyronine(T3)From tryosineTakes place in thyroid gland-follicular cellsT4 has a longer haf life than T3T4:T3 is 20:1 in blood, bound to transport protein (thyroxine-blinding globulin)A small amount is free and biologically activeT4 deiodinaes to T3 when neededIodothyrodines are the only organic molecules in the body that contain iodineT4 and T3 are lipophilic – cross the cell membrane easilyThyroid-stimulating hormone regulates their synthesis at every step. It is a glycoprotein, from the anterior pituitary. It increases basal metabolsm, heat generation and o2 consumption.

PRECURSOR: thyroglobulin

OVERVIEW: iodide anions are oxidized by thryoperoxidase (TPO) and incorporated to tyrosyl residues of thyroglobulin.

Tyrosine is converted to thryoglobuin in thyroid follicular cells. Thyroglobulin reacts with I2 to form monoiodotyrosine and diiodotyrosine (MIT/DIT).Thyroxine is formed when two molecules of DIT combine.T3 is formed when a molecule of MIT and DIT combine.

30 Intracelullar Ca 2+ distribution - calcium channels, carriers, Ca 2+ -dependent proteins (e.g.

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calmodulin) and enzymes, relations to cell functions.

Distribution:

Whole Ca2+ = 1-1.3kgIt is located in the bones (99%) and body fluis (ICF 0.9% ECF 0.1%)

Blood plasma concentration is (2.5mmol/l):50% free ionized Ca2+ BIOLOGICALLY ACTIVE32% Ca2+ bound to albumin8% Ca2+ bound to globulins10% Ca2+ bound in complexes with anions CHELATED

Ca 2+ functions: It is a bone componentSignalling substance, second messengers in transduction pathways

-cause exocytosis-muscle contraction-co-factors in blood coagulation

Stored in the SER, which keeps the cytoplasm levels low – good function in sarcoplasmic reticulum-for the release and uptake, SER membranes contain signal controlled Ca2 channels with energy Dependant Ca2+ATPase

Ca 2+ -Calmodulin: Calmodulin is a small protein found in all animal cells, which can bind 4 Ca2+ions1. Hormone binds receptor in the cell membrane2. Via G-Proteins, this has 2 actions

-mobilises intracellular Ca2+stores- opens Ca2+channels in the cell memrane

3. Activated G-protein activates phospholipase-PLC catalyses the hydrolysis of PIP2 to DG and IP3-DG activated PKC which phosphorylates enzymes-IP3 opens Ca2+channels in ER

4. Ca2+ binds to calmodulin and this complex produces physiological actions5. It activates calmodulin dependant kinases – phosphorylated intracellular proteins for a biological response

31 Calciferols (calciols) - structure, sources, transformations, effects, mechanism of action.

The calciols are several forms of vitamin D, a family of sterols that affect calcium homeostasis. Their daily requirement is 5-20ug. D-provitamins (ergostrerol and 7-dehydrocholesterol) are widely distributed in animals and plants.

Most natural foods have a low content of vitamin D3. It is present in egg yolk, butter, cow's milk, beef and pork liver, animal fat and pork skin. The most important vitamin D (D2) source is fish oil, primarily liver oil.

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7-Dehydrocholesterol

Cholesterol

An intermediate(praevitamin)

Calciol (vit. D3)

THE LIVER CELLS7,8-Dehydrogenation

Capillaries of the SKINA high-speed photolysis

λmax = 295 nm

Slow thermal conversion

Calciol is slowly released into bloodand bound to serum DBP (D vit. binding protein).

Lumisterol

Tachysterol

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The calciols are 9,10-sekosteroids, in which the ring B is opened.

Calciol (cholecalciferol, vitamin D3) Ercalciol (ergocalciferol, vitamin D2)

The effects of calciols:1. Increase absorption of Ca2+ by enterocytes 2. Regulates reabsorption and regeneration of bone tissue

In human liver, a small amount of cholesterol transforms into 7-dehydrocholesterol and from that, in dermal capillary exposed to sun radiation, calciol (cholecalciferol, vitamin D3) is formed - by of opening of the ring B(C9-C10 bond):

Calcidiol is the major circulating metabolite of calciol. Its biological half -life is rather long, approx. 20 - 30 days. The concentration of calcidiol in blood plasma informs of the body calciol saturation. Seasonal variations are observed.

25-Hydroxylation of calcidiol is inhibited by the high concentrations of calcidiol and calcitriol (feedback control), calcitonin, and the high intake of calcium in the diet.

Calcitriol has a short biological half-life. 1-Hydroxylation is stimulated by parathyrin (PTH), inhibited by calcitonin and high concentrations of calcitriol.

Calciol is an inactive precursor of calcitriol, the most potent biologically active form of vitamin D.

The hydroxylation of calciolsC-25

1αThe LIVER CELLS

25-Hydroxylation(monooxygenase, cyt P450)

The RENAL TUBULAR CELLS

1α-Hydroxylation(monooxygenase, cyt P450)

Calciol(Cholecalciferol)

Calcidiol(25-Hydroxycholecalciferol)

Calcitriol(1α,25-Dihydroxycholecalciferol)

A CALCIOTROPIC STEROID HORMONE

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32 Calcium and (inorganic) phosphate metabolism - distribution in the body, mineral deposits and soluble forms, the role of PTH, calcitriol, calcitonin.

Calcium = 1-1.3kg (99% bone, ICF 0.9%, ECF 0.1%)

Blood plasma concentration is (2.5mmol/l):50% free ionized Ca2+ BIOLOGICALLY ACTIVE32% Ca2+ bound to albumin8% Ca2+ bound to globulins10% Ca2+ bound in complexes with anions CHELATED

Hormonal control of plasma caclium concentration:PARATHYRIN – secretion regulated by plasma Ca2+ concentration: secreted in HYPOCALCEMIA.

Stimulates bone resportion through differentiation and activation of osteoclastsIn the renal tubules, Ca2+ resorption increases and HPO42- resporption decreasesIncreased calcium absorption results in the intestines

CALCITONIN – secreted by the C-cells of the thyroid gland: secreted in HYPERCALCEMIACounteracts PTH in the control of Ca metabolismInhibits bone resorptionSupports synthesis of organic matrix and mineralization of osteoidInhibits resorption of Ca2+ AND phosphates, increasing both their excretion in this way

CALCITROL – steroid hormone, from tthe kidneysStimulates resporption of Ca+ and HPO42- from the renal tubulesIncreases blood Ca2+ concentration by increased Ca2+ mobilization from boneIncreases plasma level of both ions

HypercalcemiaPlasma concentrations above 3.5mmol/lRenal functions are imparedSoft tissue calcification and renal stones develop

HypocalcemiaPlasma concentration is below 2mmol/lIncreased neuromuscular excitability and tetany (carpopedal spasms)

33 Synthesis and inactivation of catecholamines, degradation products.

Biogenic amines with a catechol group. Biosynthesis occurs in the adrenal cortex and CNS, from tyrosine.

Tyrosine hydroxylation is the rate limiting step.

Inactivation is by MONOAMINE OXIDASE (MAO). They are found in the neural tissue, gut and liver. Inactivation is by means of oxidative deamination to acidic metabolites and 3-O-methylation to metanephrines. Metabolic products of these reactions are excreted in urine as vanillylmandelic acid, metanephrine, and normetanephrine.

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Pregnenolone 17-Pregnenolone

17-Progesterone

11-Deoxycortisol

Cortisol(Hydrocortisone)

Pregnenolone 17-Pregnenolone

17-Progesterone

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34 Glucocorticoids - structure, biosynthesis, function, regulation of secretion.

Are synthesized mainly in the zona fasiculatis of the adrenal cortex.

Function:play crucial role in adaption of the organism to the state evoked by stress. They increase glucose concentration in blood by stimulating liver gluconeogenesis. They also make amino acids more easily available by suppressing proteosynthesis and supporting breackdown of proteins. Administration of high doses of glucocorticoids can evoke immunosuppressive effect, necessary after organ transplantations.Glucocorticoids have anti-inflammatory effects.

The most important glucocorticoid is Cortisol; secretion controlled by ACTH (adrenocorticotrophic hormone).

Synthesis:Cortisol – is a major glucocorticoid, synthesized from progesterone by hydroxylations at C17, 21, and 11. Secretion under basal conditions 22-70umol/day.

35 Mineralocorticoids - structure, biosynthesis, function, regulation of secretion, the renin-angiotensin system.

Synthesis occurs in the zona glomerulosa of the adrenal cortex. The zona glomerulosa doesnt express the 17-hydroxylase, so it doesnt produce precursors of gluticoids. It is the site of aldestorone production. The synthesis and secretion is controlled by Renin-Angiotensin system. ACTH influence is very weak.

Functions:- Act on the kidney to increase reabsorption of Na+ and the excretion of K+, leading to increase in BP and volume(this is effective in keeping the water mineral balance)

Cholesterol > Pregnenolone > Progesterone > Corticostreone > Aldosterone

Renin-Angiotensin System:1. Decrease in blood volume causes a decrease in renal perfusion pressure = increases renin secretion.Renin is an enzyme that catalyses the conversion of angiotensinogen to angiotensin I. Then angiotensin I > angriotensin II by angiotensin converting enzyme ACE. 2. Angiotenin II acts on zona gomerulosa to increase conversion of corticosterone to aldosterone3. Aldosterone increases reanal Na+ reabsorption, restores ECF volume and blood volume back to normal.

Renin is produced when stimulated by:Decrease in pressure in afferent arteriolesCirculating catecholeamines

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Decrease of [Na+] and [Cl-] in the tubular fluid

36 Alkali cations - distribution in various compartments, approx. daily intake and output, control of the excretion (angiotensin-aldosterone, natriuretic peptides), consequences of retention or of heavy losses of electrolytes.

Plasma cations + ECF: ICF:

[Na+] – 140mmol/l [Na+] – 10mmol/l

[K+] – 4.4 mmol/l [K+] – 155 mmol/l

[Ca2+] – 2.5mmol/l [Ca2+] – 1umol/l

[Mg2+] – 1mmol/l [Mg2+] – 15mmol/l

Daily Intake:

Na+ 500mg/d

K+ 4mg/d

Ca2+ 20-25mmol/d

Output:Ca2+ 17-25mmol/d

Angiotensin-aldosterone:Renin, angiotensin and aldosterone work together to maintain blood pressure.

Deacreased blood pressure makes kidneys release rennin by juxtaglomerular cells.

Anginotensinogen>Angiotensin I>Angiotensin II>increases production of aldestorone

Na+ and H20 retention increases, which increases blood pressure and volume

Natriuretic peptides

Atrial natriuretic peptides, ANP, are secreted by atrial myocytes. ANP acts to reduce the water, sodium and adipose loads on the circulatory system, thereby reducing blood pressure.

Secreted in response to:

- Atrial distention- Sympathetic stimulation- Increased [Na+]- Angiotensin II

ANP decreases Na+ and H2O which decreses blood pressure and volume. At the same time, it increases K+.

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Brain natriuretic peptide is secreted by heart ventricles due to excessive stretching of heart muscle cells. Aswell as decreasing blood pressure and volume, it also increases cardiac output.

37 Sex hormones (structure, biosynthesis, function, sites of secretion and their regulation, inactivation).

Testosterone (C17) – synthesised in Leydig cells in the testis.

Oestrogen and progesterone – developing follicles of the corpus luteum in the ovaries.

Adrenal Androgens – need 17a-hydroxylation

ANDROSTENEDIONE (precursor for testosterone)

TESTOSTERONE

Dihydrotestosterone and estradiol are also in the circulation, from the conversion of testosterone.

OESTROGEN:

Synthesis is stimulated by LH and FSH. The precursor is an androgen (enzyme is cytochrome P450) which is hydroxylated twice on the methyl group on C19, and then hydroxylation of C2 forms a product which gives an aromatic ring at A:

Three types are produced: estriol, estradiol and estrone.

ESTRIOL

Progesterone:

Prepares the lining of the uterus for implantation of an ovum and is also essential for the maintenance of

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pregnancy. It is also a precursor for androgens and estrogens.

Cholesterol > Pregnenolone > Progesterone > Androgens > Estrogens

It is rapidly removed from the circulation; coverted to pregnanediol and conjugated to glucunnate in liver to be excreted as urine.

PROGESTERONE

38 Neurons - components of an axon membrane and myelin, provision of energy and nutrient requirements, relationship of neurotransmitters to amino acids (a survey).

Dendrites: have receptors for neurotransmittersPerikaryon: body – have the nuclues and is the metabolic centre

Axon: for pimary active transport of Na+/K+ across the axolemma, contains voltage gated channelsAxonal transport: transport along microtubules, anterograde and reterogradeNodes of Ranvier: provides method of fastor saltatory conductionAxon terminals: synapses where neurotransmitter is released from synaptic vesicles by exocytosis

Myelin:Myelin sheaths are wrapping of glial cells around the axons. In CNS glial cells are oligodendrocytes, in PNS they are Schwann cells.

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Energy and Nutrient Requirements:Glucose is the main nutrient, in prolonged starvation KB can provide half the energy requirements. This is why impairment of consciousness is the first sympton of hypoglycemia.

Other neurotransmitters such as catecholamines are synthesized from the amino acid tyrosine which is a hydroxylate of phenylalanine.

39 Membrane potential of a neuron, depolarization and the action potential propagation. Voltage- operated and receptor-operated (ligand-gated) ion channels.

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Myelin membranes contain about 80 % lipids.

The main proteins are

- proteolipidic protein,

- the basic protein of myelin (encephalitogen),

- high molecular-weight protein

called Wolfram's protein.

the "outer“ sides

cytoplasmic sides

NeurotransmittersA large number (much more than 30) of neurotransmitters

have been described. Many of them are derived from

simple compounds, such as amino acids and biogenic

amines, but some peptides are also known to be important

neurotransmitters. The principal transporters: Peripheral neurons

– efferent

excitatory acetylcholine

noradrenaline

– afferent sensory neurons

excitatory glutamate

(Aβ fibres, tactile stimuli)

peptide substance P

(C and A fibres, nociceptive)

Central nervous system

inhibitory GABA (at least 50 %)

glycine (spinal cord)

excitatory glutamate (more than 10 %)

acetylcholine (about10 %)

dopamine

(about 1 %, in the striatum 15 %)

serotonin

histamine

aspartate

noradrenaline (less than 1 %,

but in the hypothalamus 5 %)

adenosine

neuromodulatory endorphins, enkephalins,

endozepines, delta-sleep inducing peptide,

and possibly endopsychosins.

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Neurons are irritable cells that react, after an adequate

stimulation, by formation of nerve impulses – action potentials

caused by changes in ion flows across cell membranes. Action

potential spread without decreasing along axons to the axon

terminals.

The lipidic dilayer is practically impermeable to the unevenly

distributed Na+ and K+ ions. The resting membrane potential –

70 mV on the inner side of the plasma membrane.

Sodium and potassium ion channels allow the passive passage

across the membrane:

– leakage (voltage-independent) K+ channels,

– ligand-gated Na+/K+ channel,

– voltage-operated Na+ channel, and

– voltage-operated K+ channel.

The inward flow of Na+ is the cause of depolarization (spike

potential), the following outward flow of K+ repolarization and the

refractory phase.

The original uneven distribution of ions is restored by

– Na+,K+–ATPase.

of all types are receptors cooperating with G

proteins.

-Adrenergic receptors

After binding an agonist, all types of -receptors activate Gs

proteins so

that adenylate cyclase is stimulated, cAMP concentration

increases,

and proteinkinase A is activated. Particular types differ

namely in

their location and affinity to various catecholamines:

1

are present in the membranes of cardiomyocytes,

2

in the smooth muscles and blood vessels of the

bronchial stem,

3

in the adipose tissue.

2

-Adrenergic receptors

The effect is quite opposite to that of -receptors, binding of

catecholamines results in the interaction with Gi

protein,

decrease in adenylate cyclase activity and in cAMP

concentration.

1

-Adrenergic receptors

activate Gq

proteins and initiate the phosphatidylinositol

cascade by

stimulation of phospholipase C resulting in an increase

of

intracellular Ca2+ concentration and activation of

proteinkinase C.

Adrenergic receptors

Adrenergic synapseNeurotransmitter of most postganglionic sympathetic neurons

is noradrenaline.depolarization wave

Ca2+

adrenergic receptors in

membranes of the target cells

DA -hydroxylase

synaptic vesicles

(axonal transport)

NORADRENALINE

presynaptic

adrenergic

receptors

mitochondrial

monoamine oxidase

partial reuptake

Varicosities of the postganglionic sympathetic

axons are analogous to the nerve terminals.

extracellular COMT

(catechol O-methyltransferase)

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40 Adrenergic synapse (release and inactivation of the transmitter, the types of adrenergic receptors, signal transduction).

Adrenergic synapses release catecholamines by endocytosis due to increased conc. of Ca 2+ in ICF !

Inactivation of the transmitters is done:- Acetylcholine => is cleaved by acetylcholinesterase - norepinephrine and epinephrine are taken upp by thepostsynaptic/presynaptic membrane => reuptake.

41 Cholinergic synapse (biosynthesis of the neurotransmitter and the release of it, two principal types of acetylcholine receptors and mechanisms of their function).

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Cholinergic synapse

ACETYLCHOLIN

membrane-bound

acetylcholinesterase

choline

acetyl-CoA

ATP

depolarization waveCa2+

Na+

choline acetyltransferase

(by axonal transport)

acetylcholine receptors

acetateIncrease in intracellular [Ca2+] activates Ca2+-calmodulin-dependent protein- kinase that phosphorylates

synapsin-1; its interaction with the membrane of synaptic vesicles initiates their fusion with the presynaptic

membrane and neurotransmitter exocytosis. The membranes of vesicles are recycled..

reuptake

At neuromuscular junctions, the arrival of a nerve impulse releases about

300 vesicles (approx. 40 000 acetylcholine molecules in each), which

raises the acetylcholine concentration in the cleft more than 10 000 times.

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Acetyl choline is the neurotransmitter. Acetyl choline formation takes place in the cytoplasm of the presynaptic axon.

Choline + Acetyl Co-enzymeA Acetyl choline

Inactivation of acetyl choline by acetylcholine esterase is in the synaptic cleft.

Cholinergic synpase:Depolarisation causes intracellular Ca2+ concentration to increaseThis activates calcium-calmodulin dependant protein kinase > phosphorylates synapsin-1This interacts with synpatic vesicles, initiates there fusion with the presynaptic membrane and neuroT exocytosisMembranes of vesicles are recycles

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Cholinergic synapse

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Nicotinic receptors are ligand-gated ion channels, for Na+ influx on normal action potential producing structures i.e. nerves of muscles. neural nicotinic cholinergic receptors for Ca2+ permeability in synaptic facilitation and learning.

42 Acetylcholinesterase and its inhibitors (examples of organophosphate insecticides, typical signs of toxic effects, the first aid - the counteractive alkaloid).

The effect of organophosphates is based on the fact that they block covalently the enzyme acetylcholine esterase, which catalyzes the hydrolytic breakdown of acetylcholine in the synaptic gap. [Acetylcholine is not sufficiently broken down; it cumulates and causes long-term stimulation of the receptors in the postsynaptic membrane. Therefore organophosphate poisoning is viewed as a long-term stimulation of the motor neurons and the stimulation of the parasympathetic nervous system.]

Inhibitors:

Principle – esterification of serine hydroxyl in the active site of the enzyme

1) Reversible: Carbamates2) Irreversible: Organophosphates (form a covalent bond with enzyme)

Signs of toxic effects:S - salivationL - lacrimation

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Nicotinic cholinergic receptors

are acetylcholine-operated Na+/K+ channels

(see picture 11);

in the peripheral nervous system, they occur

– in the dendrites of nearly all peripheral

efferent neurons

(including adrenergic

neurons), and

– at neuromuscular junctions ion the

cytoplasmic

membranes of skeletal muscles.

exist in two principal types that are named

nicotinic and muscarinic after the two

exogenous agonists.

Muscarinic cholinergic receptors

Five types M1–5

exhibiting different functions

are known.

In the peripheral tissues innervated by the

parasympathetic system,

receptors M1

predominate, the other types

occur mostly in CNS.

After acetylcholine has bound at muscarinic

receptors M1

, the

complex activates Gq

proteins; the

consequence - activation of the

phosphatidylinositol cascade: IP3

increases the

intracellular Ca2+

concentration, proteinkinase C is activated by

diacylglycerol.

Acetylcholine receptors

Atropin is an acetylcholine antagonist at muscarinic receptors.

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Cl–

––

––

–– –

12

2

21

In the spinal cord and the brain stem, glycine has the similar function as GABA in the brain. The inhibitory actions of glycine are potently blocked by the alkaloid strychnine, a convulsant poison in man and animals.

Inhibitory GABAA receptoris a ligand-gated channel (ROC) for chloride anions. The interaction with-aminobutyric acid (GABA) opens the channel. The influx of Cl– is the cause of hyperpolarization of the postsynaptic membrane and thusits depolarization (formation of an action potential) disabled.

The receptor is a heteropentamer(three subunit types). Besides the binding site for GABA, it has at least eleven allosteric modulatory sites for compounds that enhance the response to endogenous GABA – reduction of anxiety and muscular relaxation: anaesthetics, ethanol, and many useful drugs, e.g. benzodiazepines (hence the

alternative name GABA/benzodiazepine receptors), meprobamate, and alsobarbiturates. Some ligands compete for the diazepam site or act as antagonists(inverse agonists) so that they cause discomfort and anxiety, e.g. endogenouspeptides called endozepines.

GABA (-aminobutyric acid) is the major inhibitory neurotransmitter in CNS.

Gabaergic synapses represent about 60 % of all synapses within the brain.

GABA

mitochondrial synthesis

of GABA from glutamate

depolarization waveCa2+

GABA / benzodiazepine

receptors

uptake of GABA into glial cells

and breakdown to succinate

partial reuptake

(transporters GAT 1,2,3,4)

Inhibitory synapse

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

U – urinary incontinenceD - defacationG – GI upsetE - EmesisM – Miosis

First Aid:

Atropine: blocks the parasympatheric nervous system, both vagal effects on the heart by blocking the acetylcholine action at the muscarinic receptors.

Organophosphates => very strong nerve paralyzing poisons, which can be absorbed through the skin. The most example of toxic insecticide, commonly used in agriculture is parathion or the most toxic mevinfos.

43 Inhibitory GABAergic synapse (GABA A receptors, the effect of benzodiazepines and other ligands).

44 Retinol and its derivatives - the biological role, biochemistry of visual excitation (activation of transducin, consequences in decrease of cGMP with hyperpolarization and in decreased Ca 2+ stimulating guanylate cyclase).

Retinol (Vitamin A)Primary alcohol containing B-ionone ring and unsaturated side chain. Found in animal tissues as a retinyl ester with a long chain fatty acid.

Retinal – component of phodopsin of rod cells in the retinaAldehyde from retinol oxidation, both can be interconverted.

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Retinoic Acid – takes part in cell regulation of gene expressionIs an acid from the oxidation of retinal. It can’t be reduced in the body to give retinol or retinal.

B-carotene: is from plant food, can be oxidatively cleaved to give two molecules of retinal.

Retinoids are essential for vision, reproduction, growth and maintinance of epithelial tissues. Retinoic acid mediates most of the actions of the retinoids except vision, which is mediated by retinal.

Sources of vitamin A: CARROTS, liver, kidney, egg yolk and butter.

! Rhodopsin is found in rods (photoreceptors). It is a light sensetive chromoprotein. Opsin part contains retinal. Absorption of a photon triggers isomerisation of retinal. This leads to allosteric conformational change of rhodopsin, which binds to G-protein-TRANSDUCIN. A signal cascade follows and rods release less neurotransmitter (glutamate). Bipolar neurons register this change and transmit it to the brain for light.

In the dark, rod cells have a high concentration of cGMP (synthesized by guanylate cyclase), which binds to an ion channel to open it and allow Na+ and Ca2+ to enter, causing depolarization and release of glutamate neurotransmitter.

! Decrease of cAMP => Na+ channels closes

45 Distribution of body water, factors influencing the distribution of body water and its excretion (ADH, aldosteron, natriuretic peptides), consequences of retention or of dehydration .

Distribution of body water:

Total body water: 60% - ECF is 20% (1/3) ICF 40% (2/3)

ECF: ¼ blood plasma and ¾ interstitial fluid

Higher in new borns and adult males

Lowest in females and fat people

Factors Influencing the distribution:

AGE: highest in newborns, lowest in old femalesGENDER: higher in males, lower in femalesWEIGHT: fat has 2% water content, whereas other tissues have 73% water content (more fat=less water)

ADH (anti-diuretic hormone or vasopressin):

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from the posterior lobe of the pituitary, increases water permeability of the distal tubules and collecting duct.

Aldosterone:Decrease in blood volume causes decrease in renal perfusion pressure which causes an increase in renin secretion. Renin converts angiotensinogen to angiotensin I, and then ACE converts it to angiotensin II, which acts on the zona glomerulosa to increase conversion of corticosterone to aldosterone. Aldosterone increases renal resporption of Na+ and so increases blood volume back to normal.

Natriuretic peptides:Atrial natriuretic peptides, ANP, are secreted by atrial myocytes. ANP acts to reduce the water, sodium and adipose loads on the circulatory system, thereby reducing blood pressure.

Secreted in response to:

- Atrial distention- Sympathetic stimulation- Increased [Na+]- Angiotensin II

ANP decreases Na+ and H2O which decreses blood pressure and volume. At the same time, it increases K+.

Brain natriuretic peptide is secreted by heart ventricles due to excessive stretching of heart muscle cells. Aswell as decreasing blood pressure and volume, it also increases cardiac output.

46 Osmotic and oncotic pressure of blood plasma, plasma osmolality (values of the main parameters, empirical relations for a rough estimate of plasma osmolality) and osmolality regulation.

Osmotic pressure: hydrostatic pressure produced by a concentration gradient between two solutions on either side of a semipermeable membrane.

Oncoptic pressure: a form of osmotic pressure exerted by protein in blood plasma that tends to pull water in to the circulatory system.

Plasma osmolarity: a measure of the concentration of substrates in blood (Na+, K+, Cl-, urea, glucose, etc). The units it is measured in is ‘osmoles of solute per kg of solvent’ – mmol/kg H2O.

RANGE: 275-299 mmol/kgH2O CRITICAL VALUE: 250 mmol/kgH2O

Urine osmolarity = 500-850 mmol/kgH2O.

Osmolarity Regulation:Body osmolarity is controlled by regulating the amount of water in the body through changes in the thirst and renal

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water excretion. This controls body volume.

If Na+ is high in the body, body water will be increased to reduce the osmolarity back to normal. The body volume will then also increase.

If the body volume is too low, ADH is released which promotes water resorption in the kidneys.

Body osmolarity is sensed by osmoreceptors in the hypothalamus, which influences thirst and ADH secretion. Increase in osmolarity leads to an increase in thirst, and an increase in ADH secretion, which decreases renal water excretion.

47 Electrolyte status of blood plasma. Relation of ion concentrations to acid-base balance (buffer base and strong ion difference, anion gap).

Cations Molarity ChargeNa+ 142 142K+ 4 4Ca2+ 2.5 5Mg2+ 1.5 3 Total charge: 154

Anions Molarity Charge

Cl- 103 103

HCO3- 25 25

Proteins 2 18

HPO42- 1 2

SO42- 0.5 1

Organic 4 5 Total Charge: 154

Strong Ion Difference:

SID = [Na+] + [K+] - [Cl-] = 38-46mmol/l (proportional to buffer base of serum)

SID composition = HCO3- + HPO42- + Prot-

Strong ions don’t hydrolyse in aqueous solution.

Increased strong ion difference leads to long vomiting due to loss of Cl-.

Anion Gap:

Aproximate extent of unmeasured anionsAG = [Na+] + [K+] - [Cl-] - [HCO3-] = 12-18mmol/l

AG compostition: HPO42 + Prot- + SO42- + OA

Causes of increased AG:

- Kidney insufficiency- Diabetes, starvation- Poisoning by methanol- Lactoacidosis- Severe dehydration

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48 Transport of CO 2 in blood: p CO 2 in arterial and venous blood, [HCO3– ], carbaminohaemoglobin,

physically dissolved CO2, the ratio HCO 3- / CO 2+H2CO3 ).

There are 3 forms of CO2 transport in blood:HCO3- = 85%Protein carbamates = 10%Physically dissolved = 5% (CO2 is more soluble in blood than O2)

pCO2 of arterial blood: 4.6 – 6 kPa venous blood: 5.3 – 6.6 kPa

[HCO3-] is the only method which communicates with the external environment. It is a buffer system found in erythrocytes.

CO2 + H2O > H2CO3 (carbonic acid) > HCO3- (bicarbonate) + H+

^first step catalysed by carbonic anhydrase

[HCO3-]/[CO2+H2CO3] = 20:1

Concentration of buffer base is 20x more than the concentration of the buffer acid. It shows that it is 20x more resistant to acids.

Carbaminohaemoglobin: Hb + CO2

- A reversible reaction- covalently bound to the N-terminus of heams (not iron!)- can also bing to the amino groups on the polypeptide chains of plasma proteins

49 The acidic products of metabolism (H + -producing processes, approximate daily amounts of formed non-volatile acids, the origin of metabolic acidosis and alkalosis).

Main acidic products:Lungs = CO2 – = 25, 000 mmol/d

Kidneys = H+ (NH4+ and H2PO4) = 80mmol/d

Kidneys = HCO3- = 1mmol/d

H+ producing process:

Non electrolyte > acid > anion- + H+e.g. anaerobic glycolysis: glucose > 2 lactate- + 2H+

H+ consuming reactions:

Anion- + H+ > non-electrolyte

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e.g. gluconeogenesis from lactate: 2 lactate + 2H+ > glucose

Production of CO2:Decarboxylation reactionse.g. Oxidative decarboxylation of pyrvate > Acetyl CoA

Acidic Catabolytes:- Aerobic metabolism of nutrients > CO2- Aerobic glycolysis > lactic Acid- KB production > acetoacetic acid/B-hydroxybutyric acid- Catabolism of cysteine > sulphuric acid- Catabolism of purine bases > uric acid- Catabolism of DNA/RNA > HPO42- + H+

Metabolic Acidosis:Increased production of endogenous H+ - lactoacidosis, ketoacidosis...Intake of exogenous H+ - metabolites from methanol, administration of HCl...Loss of HCO3- and Na+ - diarrhea, burns, renal tubular disordersExcessive infusion of NaCl solution – dilution of plasma

Metabolic Alkalosis:Loss of Cl- and H+ - by vomitingIntake of HCO3- - excessive use of baking soda or alkaline mineral waterLoss of Cl- and K+ - by diureticsHypoalbuminemia – liver damage, severe malnutrition, kidney disease

50 Buffering systems in blood, blood plasma (components, concentrations), the main buffer bases in interstitial and intracellular fluids.

Three main buffering systems: pKa 6.0 – 8.0Buffer Blood Plasma RBCHCO3-/H2CO3 + CO2 50% 33% 17%

Protein/Protein-H+ 45% 18% 27%

HPO42-/H2PO4- 5% 1% 4%

TOTAL BUFFER BASES (mmol/l) 48+3 42+3 56+3

Buffer capacity depends on concentration of both components and the ratio of both components. The best capacity is when buffer base concentration equals buffer acid concentration.

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pH = pKa + log [BB]/[BA]

Hydrogen Carbonate Buffer:

This is the only buffer system which communicates with the external environment.

CO2 + H2O > (carbonic anhydrase) H2CO3 >(dissociates) H+ + HCO3-

Effective concentration of carbonic acid (mmol/l) is 0.22 x pCO2 (0.22 is the solubility coefficient of CO2)

H+ + HCO3- > H2CO3 > H2O + CO2

OH + H2CO3 > H2O + CO2

Hydrogen Phosphate Buffer:H2PO4- > HPO42- + H+ pKa = 6.8

Found in ICF, bones, and urine.

[H2PO42-] : [H2PO4-] is 4:1 in blood plasma.

Protein Buffer:H-protein > H+ + protein

Histidine is the main amino acid of blood proteins.

51 The role of the kidney and of the liver in acid-base balance.

Kidneys:They excrete acidic species (NH4

+, H2PO4-, uric acid, etc)

They reabsorb basic species (mainly HCO3-)

Ammonia Excretion:NH4

+ enter tubular cells in the form of glutamine > (glutaminase) NH4+ + glutamate

NH4+ enters the urine by the K+ channel

NH3 can freely diffuse through the tubular membrane30-50mmol/d of NH4

+ excretedOther amino acids also give NH3 (alanine, serine, Glycine, etc)

Proton Excretion:Renal tubule cells can secrete H+ even though there is a concentration gradient from the blood to the urine

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CO2 + H2O H2CO3 H+ + HCO3-

HCO3- goes back to the blood via Cl-/HCO3

- antiport or Na+/HCO3- antiport

H+ enters the urine by secondary active transport in Na+/H+ antiport

[Na+] gradient is the driving force for the proton excretion

52 Blood acid-base parameters (reference values, changes of the values in acute disturbances and in the course of their compensation).

Ph = 7.40 + 0.04pCO2 = 4.6 – 6.0 kPa

Oxygen parameters: pO2 = 12-13.3 kPa3O2 saturation of Hb by O2 is 94-99%Total Hb = 2.15-2.65mmol/l

Tissue hypoxia of any origin leas to lactic acidosis.

HCO3- 24+3mmol/lBase Excess 0+3mmol/lBB serum 42+4mmol/l (lower because it doesnt include RBC which have haemoglobin)BB blood 48+3mmol/l

Compensation: the process which occurs when one body system replaces the disturbed function of another, so that the ratio of [HCO3-] / pCO2 gets closer to normal (20:1)

Correction: the process which occurs when the disturbed system itself returns the acid-base parameters to normal.

Metabolic Acidosis:In acude disorders: [HCO3-], pH and [HCO3-]/0.22pCO2 decrease – pCO2 is normalCompensation: done by lungs via hypoventilation to reduce pCO2Correction: done by the kidneys, they increase reabosrbtion of HCO3-

Metabolic Alkalosis:In acute disorders: [HCO3-], pH and [HCO3-]/0.22pCO2 increase – pCO2 is normalCompensation: done by the lungs via hypoventilation to increase pCO2Corrction: done by the kidneys, drecrease resorption of HCO3-

Respiratory Acidosis:In acute disorders: [HCO3-], pH and [HCO3-]/0.22pCO2 normal – pCO2 is increasedCompensation: done by kidneys by proton excretion and resorption of HCO3-Correction: done by the lungs via hypervention to restore pCO2

Respiratory Alkalosis:In acute disorders: [HCO3-], pH and [HCO3-]/0.22pCO2 normal – pCO2 is decreasedCorrection : is done by the lungs via hypoventialtion to restore pCO2

53 Filtration of the plasma through the glomeruli (composition and permeability of the filtration medium, glomerular filtration rate – creatinine clearance, glomerular proteinuria).

Composition and permeability:There is a layer of fenestrated endothelial cells, which are negatively charged. They have pores with a diameter of 50-100nm. Large (Mr>60 000) and negatively charged proteins can’t pass through. Microproteins (Mr<6000 -10 000) pass through easily.

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There is then a basement membraine, made of mainly collagen. It allows free movement of electrolytes, water, and small molecules. It contains sialic acid in glycoproteins, which have a negative charge and so repulse anionic proteins.

The final layer is a layer of pedicles with slit membranes between them, the pores have a diameter of 5nm.

Glomerular Filtration Rate (GFR) – Creatinine Clearance:Is the volume of blood plasma that is completely cleared of creatinine in one second.

GFR = Vp = Vu x (Cu/Cp) Units: ml/s

Corrected GFR is clearance values normalised to a standard body surface area. Creatine excretion is proportional to the surface area of the glomeruli filtration system which is assumed proportional to the body surface area.

SA = 0.167 x √ w x h

Physiological range of GFRcor= 1.3-2.6 ml/s/1.73m2

It is age and gender dependant.

Glomerular Proteinuria:The normal glomerular barrier to plasma proteins is disrpted, proteins with molecular mass higher than 60 000 are present in the urine. Proteinuria is when there is more than 300mg of total urinary protein per 24 hours.

54 Reabsorption and secretion in the renal tubules (water, electrolytes - natriuresis, low molecular compounds – glucose, amino acids, uric acid, tubular proteinuria), the term fractional excretion E/F.

Sodium Reabsorption:Reabsorption is 95-99.5%. Aldosterone increases the Na+ reuptake, especially in the distal tubule. Atrial Natriuretic peptide will decrease the Na+ uptake.

Proximal tubule: 65% reabsorptionNa+/H+ antiportNa+/Glc antiportNa+/aa antiport

Ascending limb of loop of henele: 25% reabsorptionNa+/K+/2Cl- symport

Distal tubule: 4% reabsorption Na+/Cl+ symport

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Potassium Reabsorption:Reabsorption is 80-95%, secretion is up to 200%.

Proximal tubule: 65% reabsorptionParacellular transport

Ascending limb of loop of henele: 10-20% reabsorptionNa+/K+/2Cl- symport

Collecting Tubule: SECRETION increase by aldosterone

Chloride Reabsorption:Reabsorption is 95-99.5%Proximal tubule: 55% reabsorption

Paracellular transport

Ascending limb of loop of henele: 20% reabsorptionNa+/K+/2Cl- symport

Distal tubule: 20% reabsorption Na+/Cl+ symport

Water Reabsorption:Reabsoprtion is 70-80% mostly in the proximal tubule by aquaporins. There is high permeability in the descending limb of the Loop of Henle. The distal tubule and collecting duct have AQP-2 which are dependant on ADH. This determines the final concentration of urine.

Urea Reabsorption:In the proximal tubule 5-% is reabosorbed. The collecting duct is permeable to urea. Here urea diffuse back to interstitial fluid, the descending limb (urea recycling) and the vasa recta.

Amino acids and glycerol are reabsorbed with Na+ symport (secondary active transport).

Tubular proteinuria is when there is 150mg/g of protein in urine. It occurs when reabsorption of low Mr proteins in the proximal tubule is disrupted.

Fractional Excretion: EF = Vu/GFRPortion of water excreted into urine, from the total volume of the glomerular filtrate. Physiological range is 0.985 – 0.997. A decreased value indicates Diabetes.

55 Nitrogenous urinary constituents (substances from which they are derived, average daily excretions, the major factors on which the excreted amounts depend).

Nitrogen Compounds Metabolite Origin Excretion (mmol/day) % of total N Urea detoxification of NH3 by liver 330-600 80-90%Creatinine Creatine catabolism 5-18 3-4%NH4

+ Glutaminase + GHD reactions 20-50 3-5%Uric Acid Purine Bases catabolism 1-1.5 1-2%Free amino acids Proteolysis in tissues 4-14 1-2%

! Amount of excretion depends on the intake of proteins in the diet and utilization of nitrogen by the body.

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56 Nitrogenous low-molecular constituents of blood plasma (the parent compounds and main factors influencing concentrations of urea, creatinine, urate, ammonium, amino acids).

Amino Acids Source: dietry proteins

Urea 3-8mmol/lSource: ammonia detox in liver

AmmoniaSource: deamination of amino acids

Creatinine 70-125umol/lSource: creatine catabolism in skeletal muscles, increased in case of skeletal muscle damage

Uric Acid: 200-420umol/lSource: purine bases catabolism

57 Digestion in the mouth and in the stomach (constituents of the saliva, the gastric secretion, secretion of HCl, humoral control of hydrochloric acid output).

Saliva is excreted by the salivary glands. The pH of salive is 7.0. 1-1.5l is excreted per day. It is slightly alkaline, and contains 98% water, salts, glycoproteins and lubricants, antibodies and enzymes. Also contains amylase, lipase and lysozyme.

Gastric secretion is secreted in the stomach. The pH is 1.0 and 2-3l is excreted per day. Gastric juice is neutral or slightly basic, when HCl is added the pH becomes 1-2. Mucus protects the stomach lining. HCl denatures proteins and kills bacteria. Intrinsic factor is a glycoprotein needed for reabsorption of vitamin B12. TAG lipase cleaves fats.

Humoral Control of HCL output :

Vagal stimulation increases H+ secretion directly and indirectly. Directly by stimulating parietal cells, and indirectly by innervating G-cells to stimulate gastrin secretion, which then stimulates H+ secretion by endocrine action.

Gastrin also stimulates H+ secretion by interacting with receptors on the parietal cells. It is secreted after eating a meal.

Histamine is released from ECL (enterochromaffin-like-cells) in the gastric mucosa and diffuses to nearby parietal cells to stimulate H+ secretion.

58 The bile - formation, composition, functions of the constituents.

0.6L formel per day, with a pH of 6.9 – 7.7. It is made in the liver but stored in the gall bladder. The gall bladder bile is more concentrated than liver bile because it contains no water or salts.

Composition and Functions:WaterHCO3

- neutralizes gastric juiceCholesterol waste productPhospholipids stabilize micellar dispersion of cholesterolBile salts emulsify lipids and fat soluble vitaminsBilirubin responsible for colour

CHOLESTEROL > (7a-hydroxylase) 7a-HYDROXYCHOLESTEROL > (12a-hydroxylase)

Bile acids are made in the liver by the cytochrome P450-mediated oxidation of cholesterol. They are conjugated with

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taurine or the amino acid glycine, or with a sulfate or a glucuronide, and are then stored in the gallbladder. In humans, the rate limiting step is the addition of a hydroxyl group on position 7 of the steroid nucleus by the enzyme cholesterol 7a-hydroxylase.

Primary and secondary bile acids are absorbed exclusively in the ileum and 98-99% are returned to the liver via the portal circulation.

59 Digestion and absorption of saccharides (amylases and intestinal brush-border enzymes).

Amylase is of two kinds, salivary and pancreatic. They hydrolyze starch at their glycosidic bonds and break them down to monosaccharides and disaccharides. Disaccharides (maltase, sucrose, lactase) are found on the intestinal brush border.

Sugar specific transporters allow uptake of monosaccharides into enterocytes.

Glucose and galactose are transported by secondary active transport, against a Na+ concentration gradient, maintained by Na+K+ATPase on the basal surface of the cell. This is called glucose-Na+ symport.

Another passive transporter then transports glucose and galactose into the blood, which goes to the liver via the portal vein.

Fructose and other monosaccharides participate in carrier mediated diffusion, down their concentration gradient.

If the meal has a high concentration, then some fructose and other monosaccharides remain in the intestinal lumen and can act as substrated for bacterial fermentation.

60 Digestion and absorption of lipids from the GIT (incl. chylomicrons, the fate of them).

Triacylglycerol and phospholipids are hydrophobic, and so they need to be hydrolysed and emulsified to micelles before absorption. Micelles also carry fat soluble vitamins and cholesterol.

Hydrolysis of TAG by lingual and gastric lipases makes FFA and glycerol. Bile salts emulsify products of lipid digestion in to micells and liposomes. Miceslles are soluble, so products of digestion can be transported into the intestinal lumen. Long chain fatty acids are absorbed and TAG are reformed. Chylomicrons transport the TAG to the blood via the lymphatic system which enters the blood stream at the thoracic duct.

61 Proteolytic enzymes of the digestive tract (secretion, activation, specificity), absorption of amino acids and peptides.

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There are two main classes of proteolytic enzymes, with different specificities for the amino acid forming the peptide bond to be hydrolysed.

Endopeptidases:Hydrolyse peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, giving a large number of smaller fragments. E.g. Pepsin (stomach), elastase, trypsin, chymotripsin (pancreas).

Exopeptidases: hydrolysis of peptide bonds one at a time, from the ends of the polypeptide.

Carboxypeptidases release amino acids from the free carboxyl terminal. Aminopeptidases release amino acids from the amino terminal.

In the pancreas:Proteolytic enzymes are sereted as inactive ZYMOGENS (proenzymes). The active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific bond. E.g. pepsinogen > pepsin, Trypsinogen > trypsin.

The generation of trypsin leads to the activation of other proenzymes. Enteropeptidase activates trypsin.

Individual amino acids groups have group-specific amino acid transporters. Some transport amino acids into enterocytes, in co-transport with Na+ by secondary active transport. Others participate in facilitated diffusion. Di/tripeptides enter the brush border of the intestinal mucosa cells where they are hydrolysed to amino acids and enter the hepatic portal vein.

62 Biochemistry of large intestine.

Main Roles:Absorption of H20 and electrolytesPropulsion of contents to the anorectal region

Absorption is stimulated by short chain fatty acids which are produced by anaerobic metabolism of dietry fibre by bacterial enzymes. They are absorbed by passive diffusion.

Bilirubin is broken down to urobilinogens by the enzymes of the intestinal bacteria. Most is absorbed from intestine, and eliminated by the liver. Some enters the urine (4umol/d) and some enters the faces.

When water is absorbed, the chime gets thicker. Chyme can pick up cellular debris and other waste products.

63 Collagen and elastin (structural features, biosynthesis of collagen).

Collagen exists in all connective tissue. It is a glycoprotein. 19 collagen types are known. All collagen types include the characteristic triple helix.

Collagen 1 = skin, bones and tendons.

Collagen 2 = hyaine cartilage of joints.

Collagen 3 = skin, aorta, uterus.

Collagen 4 = basement membranes.

The secondary structure is due to a high content of prolin and hydroxyproline, which leads to a steep left-handed helix with 3.3 amino acids per turn. They are held together by hydrogen bonds.

Synthesis of collagen:Intracellular: the formation of procollagen from 3 chains (triple helix). They are transported to the golgi where they

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form vesicles, which are released by exocytosis.

Extracellular: procollagen is converted into tropocollagen by removind the non-helical N- and C- terminals on both ends. Tropocollagen is aggregated into protofibirls. Interactions of protofirils with proteoglycans forms microfibrils. Maturation is by oxidation of Lysine side chains to allysine.

Degredation occurs by collagenase during starvation or inflammatory diseases.

Elastic fibres exist in arterial walls, pulmonary alveoli, skin, and ligaments. They are composed of elastin surrounded by a microfibrillar sheath that consists of fibrilin and fibromodulin. They are produced by smooth muscle cells, fibroblasts and chondrocytes. They have a large amount of cross-links so they are non-soluble. Non-polar amino acids prevail, such as Glycine, valine and proline. The chains DON’T have a regular secondary structure.

Synthesis, Ripening and maturation:This occurs only in the last phase of fetal development and is completed after birth.

Proeleastin > soluble TROPOELASTIN > insoluble ELASTIN

64 Lysine – the role of lysyl residues in connective tissue (covalent crosslinks of collagen fibrils, desmosine of elastin).

Lysine forms the cross-links in connective tissue. The initial reaction is enzymatic, the oxidative deamination of lysine side chains, occurring in the non-helical ends of the fibril (forming tropocollaens). It is catalysed by specific extracellular aminooxidase (Lysyl Oxidase).

Interchain covalent cross-links:ALDIMINE TYPE - Between aldehyde group of allysine and amino group of lysine > Aldimine (Schiff’s base). This is a rapid reaction which gives an unstable reaction. Slow hydrogenation to become more stable.

ALDOL TYPE – Two aldehyde groups of allysine react together. It is unstable and is stabilized by the elimination of water.

In elastin, merodesmosine crosslinks three side chains. Four side chains are linked by a structure called desmosine. This is extremely stabe.

65 The bone - hormonal control of bone mineralization. Biochemical markers of bone formation and of bone resorption.

Water 25% in compact boneOrganic components 30%Mineral components 40%In dry bones – organic component is 40-45% and the mineral component is 55-60%

Organic Components: Mineral Components: Collagen Type 1 HydroxylapatiteProteoglycans Calcium PhosphateOsteocalcin Calcium SaltsCitrate Alkaline salts

Bone formation:

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STIMULATED BY – parathyrin, calcitrolIHIBITED BY – androgens, oestrogen, glucocorticoids Bone resorption:STIMULATED BY – parathyrin, calcitrolINHIBITED BY – calcitonin, oestrogens

Parathyrin promotes Ca2+ release by promoting the release of cytokines.Oestrogen inhibit sstimulation of osteoclast differentiation by osteblasts.

Biochemical markers of bone formation:Catalytic Concentration of ALP in serumConcentration of osteocalcin in serumConcentration of N-teminal/C-terminal propeptides of procollagen in serum

Biochemical markers of bone resorption:Catalytic concentration of ASP in serumConcenration of C-terminal telopeptide of collagen in serumExcretion of N-terminal telopeptide of collagen in urine

66 Structure of contractile elements of skeletal muscle fibres (sarcomere, the proteins of thick and thin filaments, functions).

Muscles are parallel bundles of muscle fibres made of 2-3um thick myofibrils. Actin contains troponin and tropomyosin. Myosin contains 2 heavy chains and 4 light chains (double helix).

Mysoin has N-terminals on the heavy chains which form a globular head for ATPase activity.

Actin is made of G-actin (globular monomer) which makes a double helix called F-actin.

Tropomyosin – is a smaller double helix attached to F-actin

Troponin – bound to one end of tropomyosin

C – binds calcium

T – binds tropomyosin I – inhibits actin-myosin interactions

67 The contraction (and relaxation) cycle of skeletal muscles.

Troponin I inhibits the actin-myosin interaction.

ATP>ADP + Pi releases chemical energy which is conserved as high energy conformation of myosin head.

Ca2+ is freed from SR and binds to Troponin C.

Tropinin I is removed so myosin can bind to actin.

ADP and Pi are liberated from the myosin head and the actin filament is pulled about 10nm towards the sarcomere centre, and so contraction occurs.

Liberation of Ca2+ from Troponin C, leads to insertion of troponin I and relaxation occurs.

Calcium concentration in sarcoplasm, resting = 10-7M, contraction = 10-5M.

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68 Provision of energy for muscle contraction and relaxation (substrates and pathways depending on the intensity as well as duration of muscular work or exercise).

1. Maximal intensity (anaerobic phase, 30seconds to 2 minutes)

In the liver, glycogen is converted to glucose, which is transported in the blood to GLUT4 transporters in the woring muscle. It becomes pyruvate and then lactate. Lactate can be recycled for gluconeogenesis in the liver. Small portion of lactate becomes metabolic fuel for resting muscle and myocardium.

2. Prolonged exercise (aerobic phase)

Working muscles adapted to aerobic metabolism of glucose and FA. Resting muscle utilizes FA and KB. Glycerol from lypolysis is a substrate for liver gluconeogenesis.

Sources of ATP in muscles:

First 10 seconds: ATP and creatine phosphateAfter 30 seconds: anaerobic glycolysis: glc > lactate + 2ATPAfter 10 minutes:aerobic oxidation of glucose > 2 pyruvate > 2 acetyl CoA > 38ATPAfter 2 hours: anaerobic oxidation of FA

stearic acid > 9 acetyl CoA > 146ATPpalmitic acid> 8 acetyl CoA > 129 ATP

69 Differences in mechanisms of cardiac and smooth muscle contraction. Biochemical markers of myocardial damage.

Cardiac Muscle:Excitation-contraction couplingAction potential spreads from cell membranes to T-tubulesduring the plateau of A.P. Ca2+ enter the cellCa2+ are released from SRCa2+ binds to Troponin C, Troponin I is removed so actin and myosin can bindADP and Pi are freed, actin filament is pulled about 10nm towards the sarcomere centre

Smooth MuscleExcitation-contraction couplingNo troponinDeploarisation causes Ca2+ to flow into the cellCa2+ binds to calmodulin to form a complex which activates MYOSIN LIGHT CHAIN KINASE

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Myosin is phosphorylated and binds to actin

Biochemical markers of myocardial damage:CK-MB has increased levels at 3-6 hours after myocardial infarctionCardiact Troponin T or I increaseLD1, AST, ALT (no-longer used)Myoglobin

70 Nitroxide synthase, the origin of endogenous NO (function, explanation of organic nitrates vasodilating effect).

N=O Nitrogen Monoxide, a short lived radical (half life 5 seconds). Nitroxide synthase: An enzyme that is activated by an increase in intracellular [Ca2+]

L-Arginine > (NO synthatse) NO > guanylate cyclase activated.

Guanylate Cyclase coverts GTP to cGMP, which activates protein kinase G.

Protein Kinase G phosphorylate and inactivates MLCK = relaxation (dialation of vessel).

Endogenous sources: ArginineExogenous sources: Organic Nitrates

71 Major fractions of the plasma proteins, main components in electrophoretic fractions (albumin, haptoglobin, transferrin, examples of the acute-phase proteins, their origin and natural functions, principles of appreciating the alterations in individual plasma proteins).

Blood serum proteins have SIX main fractions. Done on a cellular acetate strip (pH8.6) by electrophoretic separation. Total serum proteins = 62-82g/l.

Migration of proteins in the electrical field depends on:- Net electrical charge- pI (isoelectric point)- molecular mass + size of protein

ALBUMIN

A1-GLOBULINS (HDL)

A2 – GLOBULINS (ceruloplasmin, haptoglobin)

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The blood clotting cascade

Extrinsic pathwayCellular injury

Intrinsic pathwayContact system

Damaged surface – contact with subendothelial collagen

(negatively-charged)

Common pathway

Thrombin

Factor XI(PTA)

XIa

Factor XII(Hageman)

PrekallikreinKallikrein

XIIa

High MW kininogen

Factor IX(AHP B)

IXaCa2+, PL

Factor X(Stuart-Prower)

Ca2+, PLXa Factor XCa2+, PL

Factor V(proaccelerin)

Va

Thrombin

Factor VIII(AHP A)

VIIIa

Thrombin

Ca2+, PL

Ca2+, PL

Ca2+, PLFactor VII(plasma protein)

VIIa

Tissue factor(tissue thromboplastin, t-TP)

Masaryk University Biochemistry II Exam QuestionsReband Ahmed & Khuram Ahmed

B1 – GLOBULINS (LDL,transferrin)

B2 – GLOBULINS

Y – GLOBULINS

Plasma Proteins: 10,000 estimated from which 22 abundant proteins represent about 99% of the total protein in human plasma.

Albumin is a major plasma protein (35-53g/l). 10-12g are produced daily. It is essential for maintaining oncotic pressure in capillaries. It has a net negative charge an acts as an important buffer base. It can also bind Ca2+ ions (50%). Hydrophobic areas of the surface transport FFA, bilirubin and hormones. Hypoalbuminaemia is when serum concentration is below 35g/l, which causes liver deseases and a decrease in oncotic pressure.

Haptoglobin is an A2-GLOBULIN which binds free haemoglobin released from erythrocytes, therby inhibiting its oxidative activity (which would damage the kidney).

Transferrin is a B1 Glycoprotein. Its serum concentration is 2.5-4.0g/l. It transports Fe3+ ions. One molecule of transferritin can bind 2 ferric ions. In iron deficiency, transferring synthesis is stimulated. In chronic alcoholism it is impared, and detection of carbohydrate deficient transferin (CDT) is a marker.

Acute Inflammatory Disease:Early = A1 and A2 increaseLater = Y increase and albumin decrease

In chronic inflammations Y increases.

Acute Phase Proteins:Positive: Ceruloplasmin, antitrypsin, and CRP.Negative: Transferrin, albumin and prealbumin,

Ceruloplasmin is a blue protein as it has 8 Cu2+ bound to it. It’s concentration is 150-600mg/l. It is an endogenous oxidant, it prevents ferroxidase activity.

Antitrypsin inhibits proteinases released from leukocytes which may attack elastin between alveoli.

Prealbumin is a tetrameric protein, and it’s function is binding of thyroxin. It is a marker of malnutrition.

72 Blood clotting cascade. Fibrinogen, transformation to fibrin, and fibrinolysis.

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Fibrinogen is a plasma glycoprotein. It has 6 chains which are covalently linked by disulfide bridges. Thrombin hydrolyzes the bonds between the polypeptide chains releasing fibrin monomers exposing binding sites leading to the spontaneous aggregation in a form producing fibrin clot. Thrombin activates factor XIII, a transglutaminase that covalently cross-links fibrin molecules by forming peptide bonds between amide of glutamine and lysine residues => stabilizing the clot.

Fibrinogen > (thrombonin) Fibrin (deposited as a fibrous network)

Thrombin:This is a serine proteinase that cleaves small peptides from fibrinogen to expose binding sites that spontaneously allow the fibrin molecules to aggregate into polymers. Covalent cros linking of fibrin by transglutaminase stabilizes the thrombus. The pathways are activated by injury to the vessel wall.

Von Willebrand factor: This is secreted by injured endothelial cells. It is a large protein which is the carrier for coagulation factor 8 and promotes platelet adhesion to collagen.

Fibrinolysis – by plasmin which digests fibrinogen, factor V, VIII, XII and prothrombin

When a clot is also forming, also plasminogen in plasm is trapped in the clot. The injured tissue slowly releases tissue plasminogen activator which starts

73 Vitamin K - the biochemical function, significance for blood clotting.

The cycle is responsible for converting (carboxylation) glutamate into Y-carboxy-glutamyl residue (Ca2+ binding centres on activated thrombocytes). This is an essential step in post-translational processing of blood co-agulation factors 8, 9 and 10 and prothrombin.

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Vitamin K metabolic cycle in the liver cells

-Carboxylation of Glu residues

that forms Gla Ca2+-binding centresis an essential step ofposttranslational processingof blood coagulation factorsVII, IX, X, and prothrombin.

The two stages of reduction of vitamin K epoxide to the hydroquinone areinhibited by coumarin anticoagulants warfarin or dicoumarol (analoguesof vitamin K) used as inhibitors of blood clotting in the treatment of thrombosis.

GlaGlu

H2O+

CO2

H2O

General Medicine 4th semester 2009