BIOCHEMISTRY II EXAM ANSWERS

Masaryk University Biochemistry II Exam Questions

Reband Ahmed & Khuram Ahmed

Biochemistry II - examination

GENERAL MEDICINE
DENTISTRY

Khuram Ahmed
Reband Ahmed

General Medicine 4th semester 2009



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 WILL
Plasmatic co-agulation factors decrease
Secretory amylase, lipase increase
Intracellular 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.




LD – Lactate Dehydrogenase
Lactate + 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 Kinase
Is 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 well- prolonged extreme
post resorption early starvation
description fed starvation starvation

Time
a 0-4 h 4-16 h 16-30 h 2-24 d over 24 d
interval

Origin of
liver glycogen gluconeogenesis
Glc in food gluconeogenesis gluconeogenesis
gluconeogenesis liver glycogen
blood
b b
all tissues all tissues
Utilization all brain, Ercs, Ercs, kidney,
muscle, ad.t. muscle, ad.t.
of Glc tissues kidney brain - limited
limited limited

Energy for Glc, ketone ketone bodies,
Glc Glc Glc
brain 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.

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/l
Phospholipids 2.5mmol/l
Triacylglycerols 1.5mmol/l
Free Faty Acids 0.5mmol/l

Classes of Lipoproteins: (increasing density, decreasing size)
Chylomicrons 85% TAG
VLDL 50% TAG
LDL 50% cholesterol
HDL 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 --> tissues
VLDL Liver Endogenous TAG from liver -- > tissues
LDL Blood Plasma Cholesteryl ester --> tissues
HDL Liver Free cholesterol --> liver

CM contains predominantly TAG = neutral molecules (without charge)
 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.

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).

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

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.

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 products
2. Synthesis of tissue/plasma proteins
3. Deamination and utilization of carbon skeleton

Digestion of proteins:
Stomach: Pepsin
Small 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 food
Egg 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 Nitrogen
Dietry proteins > AA in GIT
Transamination of AA in cells > Glutamate
Dehydrogenation 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 = output
2. 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

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 saccharides
Primary regulation of blood glucose concentration – via the glucose buffer function
Uptake of glucose and storage as glycogen
OR initiation of glycogenolysis and gluconeogenesis

Metabolism of lipids
Completion and secretion of VLDL and HDL
Ketogenesis produces ketone bodies
Secretion of cholesterol and bile acids into bile (cholesterol elimination)

Metabolism of Nitrogenous compounds
Deamination of amino acids in excess of requirements
Proteosynthesis of plasma proteins and blood-clotting factors – zone 1 periportal area
Uptake of ammonium for ureosynthesis – zone 1 periportal area
Bilirubin 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 GIT
demaination of Gln/Glu in enterocytes
In saliva from: hydrolysis of urea by oral microflora
In venous blood from: catabolism of AA in tissues
In urine from: hydrolysis of Gln

Glutamine in Muscle
Produced by proteolysis
A product of ammonia detoxification
Carrier of NH2 group to liver where NH3 is liberated

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

Glutamine in brain
Formation of glutamine is a way of amonia detoxification
Synthesis occurs mainly in astroglial cells
Glutamate decarboxylation gives GABA
GLUTAMATE + NH3 > (glutamine synthase / -H2O) > GLUTAMINE

Glutamine in Liver
Periportal Hepatocytes: Source of ammonia for ureosynthesis
Perivenous hepatocytes: a form of ammonia detoxification, released into blood to go to enterocytes and kidneys

Glutamine in kidneys
Is an energy source
Glutamine and Glutamate release ammonium ions which makes the pH of urine acidic




Multiple functions of glutamine
Synthesis of proteins
Metabolic Fuel
Source of nitrogen in synthesis of purines, pyrimidines, aminosugars
Source of glutamate for gaba synthesis
Source 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.

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 bilirubin
Hepatocellular – due to inflammation or autoimmune disease
Posthepatic – 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.

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 increases
Enzyme 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 measured
Serum catalytic concentration of alkaline phosphates
Tests 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 biotransformations
Hydroxylation (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
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

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




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 proenzyme
Active 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 subunits
Regulatory 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 proteins
Phosphorylation, 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.

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.

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 phosphodiesterase
3. Receptors with enzyme activity – granylate cyclase
4. 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 receptors
All have some common structural features:
1) extracellular parts are slightly glycosylated, have accessory binding sites for agonist
2) membrane parts: 7 a-helical segments span the membrane, connected by intra and extracellular hydrophilic
loops
3) 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

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

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.

 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 secretion
2. Increased amino acids in plasma after ingestion of proteins also increases secretion
3. Gastrointestinal horomone secretin, released after ingestion, causes anticipatory rise

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 hours
Normal <6 <11 <8
Impaired >6 >11 8-11
Diabetes 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.

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 Function
Corticotropin-releasing hormone paraventricular nuclues with ADH, stimulates anterior pit. To secrete
ACTH
Dopamine arcuate nucleus inhibits anterior pit. Secreting prolactin
Gonadotropin-releasing hormone arcuate nucleus stimulates anterior pit. To secrete LH and FSH
Growth hormone releasing hormone arcuate nucleus stimulates anterior pit. To secrete GH
Vasoprissin (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 tryosine
Takes place in thyroid gland-follicular cells
T4 has a longer haf life than T3




T4:T3 is 20:1 in blood, bound to transport protein (thyroxine-blinding globulin)
A small amount is free and biologically active
T4 deiodinaes to T3 when needed
Iodothyrodines are the only organic molecules in the body that contain iodine
T4 and T3 are lipophilic – cross the cell membrane easily
Thyroid-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 Ca2+ distribution - calcium channels, carriers, Ca2+-dependent proteins (e.g.
calmodulin) and enzymes, relations to cell functions.
Distribution:
2+
Whole Ca = 1-1.3kg
It is located in the bones (99%) and body fluis (ICF 0.9% ECF 0.1%)

Blood plasma concentration is (2.5mmol/l):
2+
50% free ionized Ca BIOLOGICALLY ACTIVE
2+
32% Ca bound to albumin
2+
8% Ca bound to globulins
2+
10% Ca bound in complexes with anions CHELATED
2+
Ca functions:
It is a bone component
Signalling 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
2
-for the release and uptake, SER membranes contain signal controlled Ca channels with energy
2+
Dependant Ca ATPase

2+
Ca -Calmodulin:
2+
Calmodulin is a small protein found in all animal cells, which can bind 4 Ca ions
1. Hormone binds receptor in the cell membrane
2. Via G-Proteins, this has 2 actions
2+
-mobilises intracellular Ca stores




2+
- opens Ca 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
2+
-IP3 opens Ca channels in ER
2+
4. Ca binds to calmodulin and this complex produces physiological actions
5. 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.

≡

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

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

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):

Cholesterol THE LIVER CELLS
7,8-Dehydrogenation
Lumisterol

Tachysterol

7-Dehydrocholesterol
Capillaries of the SKIN
A high-speed photolysis
λ max = 295 nm

Slow thermal conversion
An intermediate
(praevitamin)

Calciol is slowly released into blood Calciol (vit. D3)
and bound to serum DBP (D vit. binding protein).




Calciol is an inactive precursor of calcitriol, the most potent biologically
active form of vitamin D.
The hydroxylation of calciols
C-25

The LIVER CELLS The RENAL TUBULAR CELLS 1α
25-Hydroxylation 1α-Hydroxylation
(monooxygenase, cyt P450) (monooxygenase, cyt P450)

Calciol Calcidiol Calcitriol
(Cholecalciferol) (25-Hydroxycholecalciferol) (1α,25-Dihydroxycholecalciferol)
A CALCIOTROPIC STEROID HORMONE

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.

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):
2+
50% free ionized Ca BIOLOGICALLY ACTIVE
2+
32% Ca bound to albumin
2+
8% Ca bound to globulins
2+
10% Ca 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 osteoclasts
In the renal tubules, Ca2+ resorption increases and HPO42- resporption decreases
Increased calcium absorption results in the intestines

CALCITONIN – secreted by the C-cells of the thyroid gland: secreted in HYPERCALCEMIA
Counteracts PTH in the control of Ca metabolism
Inhibits bone resorption
Supports synthesis of organic matrix and mineralization of osteoid
Inhibits resorption of Ca2+ AND phosphates, increasing both their excretion in this way

CALCITROL – steroid hormone, from tthe kidneys
Stimulates resporption of Ca+ and HPO42- from the renal tubules
Increases blood Ca2+ concentration by increased Ca2+ mobilization from bone
Increases plasma level of both ions

Hypercalcemia
Plasma concentrations above 3.5mmol/l
Renal functions are impared
Soft tissue calcification and renal stones develop

Hypocalcemia
Plasma concentration is below 2mmol/l
Increased 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.

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 aldosterone
3. 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 arterioles
Circulating catecholeamines
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+.

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
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 neurotransmitters
Perikaryon: body – have the nuclues and is the metabolic centre

Axon: for pimary active transport of Na+/K+ across the axolemma, contains voltage gated channels
Axonal transport: transport along microtubules, anterograde and reterograde
Nodes of Ranvier: provides method of fastor saltatory conduction
Axon 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.

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.

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

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

Inactivation of the transmitters is done:
- Acetylcholine => is cleaved by acetylcholinesterase
- norepinephrine and epinephrine are taken upp by the
postsynaptic/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).
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 increase
This activates calcium-calmodulin dependant protein kinase > phosphorylates synapsin-1
This interacts with synpatic vesicles, initiates there fusion with the presynaptic membrane and neuroT exocytosis
Membranes of vesicles are recycles

+
Nicotinic receptors are ligand-gated ion channels, for Na influx on normal action potential producing structures i.e.
nerves of muscles.
2+
 neural nicotinic cholinergic receptors for Ca 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: Carbamates
2) Irreversible: Organophosphates (form a covalent bond with enzyme)

Signs of toxic effects:
S - salivation
L - lacrimation
U – urinary incontinence
D - defacation
G – GI upset
E - Emesis
M – 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 (GABAA receptors, the effect of benzodiazepines and other
ligands).

Inhibitory GABAA receptor
is 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 thus
its depolarization (formation of an action potential) disabled.
Cl– The receptor is a heteropentamer
(three subunit types). Besides the
1 2 binding site for GABA, it has at least
2 1
eleven allosteric modulatory sites for
compounds that enhance the response
2 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 also
barbiturates. Some ligands compete for the diazepam site or act as antagonists
(inverse agonists) so that they cause discomfort and anxiety, e.g. endogenous
peptides called endozepines.
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.




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 Ca2+
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 retina
Aldehyde from retinol oxidation, both can be interconverted.

Retinoic Acid – takes part in cell regulation of gene expression
Is 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
+ 2+
channel to open it and allow Na and Ca 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 females
GENDER: higher in males, lower in females
WEIGHT: fat has 2% water content, whereas other tissues have 73% water content (more fat=less water)
ADH (anti-diuretic hormone or vasopressin):
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
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 Charge
Na+ 142 142
K+ 4 4
Ca2+ 2.5 5
Mg2+ 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 anions
AG = [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

48 Transport of CO2 in blood: pCO2 in arterial and venous blood, [HCO3–], carbaminohaemoglobin,
physically dissolved CO2, the ratio HCO3- / CO2+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
e.g. gluconeogenesis from lactate: 2 lactate + 2H+ > glucose

Production of CO2:
Decarboxylation reactions
e.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 disorders
Excessive infusion of NaCl solution – dilution of plasma

Metabolic Alkalosis:
Loss of Cl- and H+ - by vomiting
Intake of HCO3- - excessive use of baking soda or alkaline mineral water
Loss of Cl- and K+ - by diuretics
Hypoalbuminemia – 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 – 8.0
Buffer Blood Plasma RBC
HCO3-/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.

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 membrane
+
30-50mmol/d of NH4 excreted
Other 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
+ -
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 + 0.04
pCO2 = 4.6 – 6.0 kPa

Oxygen parameters:
pO2 = 12-13.3 kPa
3O2 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/l
Base Excess 0+3mmol/l
BB 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 normal
Compensation: done by lungs via hypoventilation to reduce pCO2
Correction: done by the kidneys, they increase reabosrbtion of HCO3-

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




Respiratory Acidosis:
In acute disorders: [HCO3-], pH and [HCO3-]/0.22pCO2 normal – pCO2 is increased
Compensation: 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 decreased
Correction : 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.
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% reabsorption
Na+/H+ antiport
Na+/Glc antiport
Na+/aa antiport

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

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

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

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

Collecting Tubule: SECRETION increase by aldosterone

Chloride Reabsorption:
Reabsorption is 95-99.5%
Paracellular transport

Ascending limb of loop of henele: 20% reabsorption
Na+/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/GFR
Portion 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.

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/l
Source: ammonia detox in liver
Ammonia
Source: deamination of amino acids

Creatinine 70-125umol/l
Source: creatine catabolism in skeletal muscles, increased in case of skeletal muscle damage
Uric Acid: 200-420umol/l
Source: 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 B 12. 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:
Water
-
HCO3 neutralizes gastric juice
Cholesterol waste product
Phospholipids stabilize micellar dispersion of cholesterol
Bile salts emulsify lipids and fat soluble vitamins
Bilirubin 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
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.


BIOCHEMISTRY II EXAM ANSWERS

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