1 - Inner environment + blood

Question 1

1. Milieu Interieur

Answer 1

-Milieu Interieur:
•Cells of the living organism can only work in constant environment.
•This environment is called the “milieu interieur”, by Claude Bernard.
•The living organism does not live in an outer environment but in its own fluid environment.
-Homeostasis: the maintenance of a constant internal environment.
•Important in both unicellular and multicellular components of the organism.
•Isovolemia: Constant volume
•Isotonia: Constant osmotic pressure
•Isoionia: Constant ion composition
•Isohydria: Constant pH
•Water is the most important component in the body. Acts as a solvent, medium for reactions, source of osmotic forces and maintains compartment and form.

Question 2

1. Compartments

Answer 2

•The body is divided into anatomical spaces and compartments.
•They are separated by barriers:
o Cell Membrane
 Permeable for water without restriction
 Selective for every other substance
o Capillary Wall
 Retains colloids
 Permeable for all other substances
-Total Body Water (TBW) = Extracellular Compartment + Intracellular Compartment
-Extracellular compartment (33%), divided into subcompartments:
•Intravascular: plasma water, blood cells
•Interstitial: soft tissues, fibrous CT, bone tissue
•Transcellular
o Sep. from other compartments by epthelial cell layers
o Synovial fluid, aqueous humour, glandular discharges, content of urinary bladder, GI tract, (Rumen)
-Intracellular Compartment ( 66%):
•Considered to be uniform, in spite of the fact that it consists of many small compartments

Question 3

1. Changes of water compartments

Answer 3

-Major rules of compartments
•Keep IC compartment isosmotic and isovolemic
•Osmolarity of EC and IC compartment equalized in few min
•Water moved by osmotic (and hydrostatic) forces.
•Cell membrane: main barrier of substance movement.
-Dehydration (hypovolemia)
•Isoosmotic hypovolemia: haemorrhage, burn, vomiting, diarrhoea
•Hyperosmotic hypovolemia – hydropenia: decreased intake; incr. loss, increased evaporation; diabetes insipidus
•Hypoosmotic hypovolemia; decreased salt intake, primary salt loss: intensive sweating, salt loss through the kidneys; hypoadrenocorticalismus.
-Hyperhydration (hypervolemia)
•Isoosmotic hypervolemia – oedema; overdosed physiological saline
•Hypoerosmotic hypervolemia: hyperosmotic fliud intake per os or parenterally
•Hypoosmotic hypervolemia: exaggerated water uptake; water retention in kidney
-Time factors of the regulation
•IC and EC osmolarity: balance in few min. by movement of fluids and osmotically active substances.
•IC and EC isoosmosis: formed in 30 min.
•Isovolemia: restored in a couple of days. Volume shifts can be tolerated much better than shifts in osmolarity.

Question 4

2. Volume quantification of fluid compartments

Answer 4

-Dilution principle: Measuring EC compartment, 2 phases are observed:
1. Quickly equilibriating space: Substance flow is fast, equilibriates within 0.5-1hour
*Blood plasma, interstitium of soft tissues and lymph
2. Slowly equilibriating space: Completely uniform distribution develops only 8-10 h later
*Bone and transcellular space
-Stewart dilution principle: used to quantify volume
-Stewart principle in the living organism: conc. of indicator decr. constantly, indicator conc. should be calculated at administration: V=S/C0
-Assessmeent of TBW:
•Based on dilution principle: 3H2O, 2H2O, Antipirin, Urea, Tiourea
•Quantifying density in vivo
o Weigh the mass (m)
o Quatify volume (V)
o Calculate density (m/V)
•Lean Body Mass: has a constant 73% water content while fat has a 10% water content
•Calculating density: Suitable for estimating the composition of the body
•New method: Total Body Electrical Conductance measures fat content with high precision.
-In EC space, fluid distributes at different rates:
•Fast Distribution: Shows V of soft tissues
•Slow Distribution: V of soft tissues and fibrous tissues
•Late Distribution: V of bone tissues, fibrous tissues and soft tissues
-Quantification of the Intravascular Compartment:
*Plasma: evans blue or 125 Iodine
*Blood: marked RBC
-Quantification of IC Compartment: IC = TBW – EC
-Quantification of Interstitial Fluid Compartment: ISF = EC – Intravasal volume

Question 5

3. Factors influencing the EC and IC compartments

Answer 5

•Osmotic Pressure
o Measured in kPA
o P=CRT (P= Osmotic Pressure, C= Conc of dissolved substance, R= General gas constant, T= Temperature)
o P=rCRT (r= Reflection coefficient (r=1, membrane is semipermeable))
• Molarity
o Measured in mmol/l
o 1 mol dissolved anelectrolite in 1L = P of 2.27MPa
• Osmolarity
o 1 osmol/L = 6x1023 dissolved substance/L
• Molality
o Measured in mol/kg
• Osmolality
o Measured in 1osmol/kg
o Common index number for the osmotic effect of electrolites and anelectrolites
• Oncotic Pressure
o P maintained by proteins and colloid particles

Question 6

3. Anelectrolite and electrolite components of the blood plasma

Answer 6

-Anelectrolite:
o Glucose 5 mmol/L
o Urea 3-10 mmol/L
o NPN 15-25mmol/L
-Electrolite: Na+, K+, Mg2+, Ca2+, Cl-, HCO3-, protein, P, organic acids
• Osmolality calculated on the basis of total electrolyte concentration: 280mmol/kg
• Osmolality calculated by freezing point depression: 300mmol/kg
• Difference is the non-ionic osmolality

Question 7

4. Functions of plasma proteins

Answer 7

• Maintaining oncotic pressure
o Albumin: 80% responsible
o Keeps water in circulation
• Transport functions of albumin
o Fatty acids, Bilirubin, Hormones, Vitamins, Metal ions
•Transport functions of globulins
o Transferrin, Haptoglobin, Transcortin, Thyroxin Binding Globulin, Transcobalamin, Lipoproteins
•Lipids bind to proteins forming lipoproteins
o VLDL, LDL, IDL, HDL
•Blood sedimentation
o Clinical parameter
o In case of infections, acute phase proteins appear in the plasma.
o Bind to surface of RBC, reducing the charge, causing less repulsion bw RBC and thus sedimentation becomes faster.
•Buffer
o Plasma proteins: responsible for 7% buffer capacity of blood, 15% buffer capacity of plasma
•Blood clotting
o Circulate in intravasal compartment (except for Ca2+) as inactive precursors
o Also responsible for anticoagulation and fibrinolysis
•Immunity
o Igs, proteins of non-specific immunity, signal proteins and peptides
•Enzymes in the plasma
•Protein metabolism

Question 8

4. Fractination of plasma proteins

Answer 8

•Paper electrophoresis
o Only albumin and fibrinogen can be sep. with this method. The rest of the proteins can be found in the globulin fraction.
•Gel electrophoresis
o Sep. into albumin, and globulins
•Immunoelectrophoresis
o Ab distributed in a gel poured on a sheet of glass develops precipitation arcs with the Ag in the electric field
•Ion Exchange chromatography
o Sep proteins on the basis of their charge
•HPLC - High Pressure Liquid Chromatography
o Divides proteins in a solid phase column under high pressure perfusion
•Ultracentrifugation
o Spinning tubes at high G to sep macromolecules according to their sedimentation constants
•Gel-filtration
o Smaller molecules have to pass through the polysaccharide beads and hence move slower than bigger beads which just bypass the beads.
•Affinity Chromatography
o One covalently binds a specific Ab formerly produced against the protein to the granules of the solid phase. The protein will then be selected from the mixture by a special recognising system

Question 9

4. Changes of plasma proteins

Answer 9

• Hypo and Hyperproteinemia: Brought about by starving, kidney disease
• Dysproteinemia: Ratio changes
• Paraproteinemia: Pathological proteins appear
• Defect-proteinemia: Lack of some of the fractions

Question 10

5. Interstitial fluid (ISF); transport through the capillary wall

Answer 10

-Two important factors determining the formation of ISF are:
1. Transport through the capillary wall
o Water, electrolites and anelectrolites with small molecular weight
o Protein movement is restricted, only a small amount of it can get through, mainly by pinocytosis and exocytosis.
o Protein conc of ISF is high due to proteins being large enough that they cannot return to the capillary.
o Capillary wall is a considerable barrier for colloids. Transported with help of specific carrier systems
o Special capillaries such as sinusoids in the liver are permeable for proteins.
2. Forces determining transport:
o Diffusion, osmotic conditions, electric forces and hydrostatic forces
• Diffusion is the most important force to ensure transport of substances.
• If movement of some component is restricted bw two compartments, the conc of the diffusible ions will be different in the two compartments after balance develops.

Question 11

5. Interstitial fluid (ISF); measurement and composition of ISF

Answer 11

-GibbsDonnan balance:
•The diffusable ion conc in the intravasal compartment and the interstitium may hardly differ.
-Measurement of ISF: ISF = EC – Intravasal volume
-Composition of ISF: plasma/ISF
*Na+: 140/135
*K+: 4/4
*Mg2+: 1.5/1.5
*Ca2+: 1.25/1.25
*Cl-: 110/114
*HCO3-: 27/28
*Protein filtrated 0.6 g/l, finally 20-30 g/l

Question 12

6. Physiological role of the blood

Answer 12

-Blood:
•Lliquid CT consisting of 90% water
•Consists of blood plasma and suspended cellular elements
•Can be sep into a liquid phase and corpuscular elements
•Plasma contains fibrinogen – collected from anticoagulated blood
•Serum does not contain fibrinogen–collected from normal blood
-Physiological Role of Blood:
•Transportation-primary function
o Gases, nutrients, metabolites, information, heat
•Important buffer
*Bicarbonate – 53%
*Non bicarbonate – 47%
•Takes part in defence and reacts after vessel injuries
•Homeostasis: Isovolemia, Isotonia, Isoionia, Isohydria

Question 13

6. Definitions in blood volumes

Answer 13

•Hematocrit: Shows the proportion of corpuscular elements relative to the whole volume.
o Important diagnostic parameter.
o Average value is around 40% or 0.4.
•Sedimentation Rate: how quickly erythrocytes settle
o using anticoagulated blood put in a standard size tube.
o The thickness of the plasma layer formed on the top of the tube is measured in certain time periods.
•pH of the Blood: 7.35-7.45
o Extremely stable
o Can withstand fluctuations for short periods 7.1-7.6

Question 14

6. Changes in blood volumes

Answer 14

• Abnormal blood volume:
• Hypovolaemia: decr.; blood and plasma loss together
• Hypervolaemia: incr.; plethora, excess transfusion, permanent exhausting physical work
• Abnormal red blood cell amount:
• Polycythaemia: high conc. of RBC (erythrocytosis)
• Oligocythaemia: deficiency of RBC (anaemia)
• Anhydraemia: lack of water in blood. Thirst.
• Hydraemia: incr water content of the plasma. Excess water intake. Infusion.

Question 15

7. Red blood cells

Answer 15

=Erythrocytes
-Most common blood cell
-No mitochondria
-Mammals: no nucleus
-The vertebrate’s principal means of delivering oxygen (O2) to tissues
-Average lifetime: 120 days (Cattle, Swine: 60, Birds: 30)
-Hemolysis: leakage or disruption of blood cells
•RBC can undergo osmotic hemolysis.
o RBC in isotonic solution: Cell does not change
o RBC in hypotonic solution: Water flows into cell, it swells up - bursts
o RBC in hypertonic solution: Water leaves cell and it shrinks
•Adapt to these changes easily-osmotic resistance
•Hemolysis can be brought about by:
o Phys effects: freezing, dissolving, shaking, shocking
o Chem effects: acids, liposolvents, surface tension reducers
o Toxins: bacterial, snake, plant

Question 16

7. The haemoglobin

Answer 16

•Pigment colouring RBCs
•Transport of blood gases and forming of blood’s buffer capacity.
•Synthesis requires Fe, Cu, Co, Mn, Zn, Pyridoxin B6 among others.
•The haem: a ferro-protoporphyrine.
o In the porphyrine base, iron has 4 coordination sites
o The two other valences involved are bound to the imidazole ring of the protein under and over the porphyrine base.
o Only the iron atom can bind oxygen reversibly – oxygenation
o Oxygenated derivative: methaemoglobin. Binds oxygen irreversibly.
•Structure of globin: determines characteristics of oxygen binding. Allows allosteric stimulation and is able to bind new oxygen even stronger after accepting the former one.
•Sickle cell anaemia: glutamine in the 6th position of the beta chain instead of alanine.
•Age effect: HB-F has a smaller affinity to 2,3-DPG than Hb-A, due to its aa. sequence.
•Hb-A = 2 alpha + 2 beta chains
•Hb-F = 2 alpha + 2 gamma chains
-Haemoglobin amount: 120-180 g/L - 35%
-Haemoglobin as a buffer: HHb/Hb-
•Hb- anion, important buffer bases in blood
-Degradation: phagocytes convert the HEM part to bilirubin.

Question 17

8. Human and animal blood groups: blood groups

Answer 17

-Blood groups: groups of complex Ags appearing on the surface of blood cells.
•Appearance is ensured by known genetic code, encoded on the same locus.
•Can be used in parentage testing, prediction of certain production traits, selection and prevention of certain illnesses.
•Hemagglutinogens: compounds on the surface of RBCs, forming Ags - initiating production of blood-agglutinating-Ab
•Hemagglutinins: Abs directed against blood group Ags
•Neutral hemagglutinins: Abs present without previous immunization. Agglutinate RBC expressing the corresponding Ags.
•Artificial hemagglutinins: Abs formed only after previous immunization.
•Hetero hemagglutinins: agglutinates RBC of other species
•Iso hemagglutinins: agglutinates RBC of same species
•Formation of hemagglutinins: early immunization postnatally
•Reaction bw RBC and plasma Abs occur in two forms:
1. Agglutination (most typical in human)
2. Hemolysis (most typical in animals)
o Can occur together or alone
•Incompatibility: If plasma of recipient reacts with RBC of the donor

Question 18

8. Human blood groups

Answer 18

•15 types
•Known blood types: ABO, Rh, MNSs, utheran, Kell, Duffy, Kidd.
-ABO blood group system:
•H-Ag encoded by H-gene forms basic structure for ABO-system
•If ABO is missing there is no blood type: Bombay group
•If there are no additional groups synthesized (A or B), the 0 group is the default type.
•A antigen: Formed by N-acetyl-galactose-amine transferase enzyme
•B antigen: formed by galactosyl-transferase enzyme
•0 antigen: suppresses the production of N-acetyl-galactose-amine transferase enzymes. Accordingly no A or B blood group is formed and the 0 group appears.
•AB0: IgM type Abs: Form against Ags entering blood after birth, if similar structures are not already present
•AB0 blood type reactions:
o Type of RBC belonging to uknown blood group can be determined by agglutination reaction
-Rhesus blood group system (Rh +/-):
•1st pregnancy:
o A Rh-negative mother can be immunized by blood cells of her own Rh-positive foetus
o The forming Ab is IgG, crosses the placenta easier
o First foetus gives no damage because the foetus RBC usually gets into mother´s body only via micro-injuries during delivery
• 2nd pregnancy:
o Small amount of RBC entering mothers sensitized immune system stimulates production of IgG Abs that dissolves foetal RBC and causes erythroblastosis foetalis
-Rh-incompatibility:
o When a Rh-neg recipient get RBC from Ph-pos donor. The recipient is immunized and the IgG Ab dissolves RBC of recipient.
o Especially 2.blood transfusion can cause significant damage.

Question 19

8. Animal blood groups

Answer 19

•More systems and many RBC-antigens, than humans.
•Blood transfusion easier for animals:
o Only few naturally prod. Abs against RBC-Ags
o Ags in both RBC and plasma. In case of transfusion the Abs is netralized in the plasma and it´s RBC-dissolving capacity diminishes.
•Exact blood type mapping only possible by developed specific anti-sera against RBC-antigens.
•The goal of determination is production of Abs that only reacts with C-blood type Ag, altough we have no specific Ag against the single C-group.
-Chimeras: individuals with their own and their twins Ag structure
-Freemartinism: female animal, usually calf, born as the twin of a male animal and is sterile because of having abnormal internal reproductive organs.
•The testosterone of the male twin during embryonic life inhibits formation of the female twin
-Production traits: E.g. meat quality caused by H-Ag of swine
-Blood Transfusion
•Substitution of lost RBCs and assuring normal gas transport.
•Large animals: a small amount is first transfused. If there is no sign of incompatibility the whole amount is transfused.
•Small animals: three drop test must be always completed; 1 drop of donor blood cells, 1 drop of recipient’s plasma and 1 drop of physiological sodium chloride solution. If there is no hemolysis or agglutination, the transfusion can be completed without the complete knowledge about the blood groups.
•For cat as a life saving act blood of a donor dog can be transfused. Cat blood can´t be used for dogs.

Question 20

9. Neutrophil granulocytes

Answer 20

-Most abundant type of granulocytes and WBC
•Diameter: 10-14 μm
•Nucleus: stronly segmented. Depends on maturation; mature: 3-5 segments, over matured: 5-10 segments
•Granules/cytoplasm: small purple-red granules
•Enzymes: lysosime, myeloperoxidase, superoxide-dismutase, acidic and alcalic phosphatase, nuclease
•Most important element of cellular immunity: effective phagocytosis
•Lifetime: circulation 6-7 hours, tissue 2-3 days
•Circulation fraction: small
•Extravascular fraction: BM, spleen.
-Migration: neutrophils are able to actively move/migrate
•Movement is based on building up and dissociation of actin chains. Migration by philopodium.
-Phagocytosis: can take up bigger particles and engulf molecules.
•If specific and non specific factors bind a foreign body, the phagocyting ability incr 100x
•Opsonisation: some natural substances in the blood plasma can bind foreign Ags similarly to Igs. They bind with neutrophil gran. and significantly stim phagocytosis.

Question 21

9. Basophil granulocytes

Answer 21

•Rarest of WBC
•Diameter: 9-19 μm
•Nucleus: slightly granular, usually horse-shoe/S-form.
•Granules: many, big, pruple basophil granules, that contain histamine, serotonin and heparin besides the enzymes
•Enzymes: Myeloperoxidase, hyaluronidase
•Small circulation fraction: no phagocytosis, almost no endocytosis.
•Extravasal fraction: most cells can be found in the tissues
•Loosen tissues: stimulate elimination of antigens
-Degranulation: bind the ready IgE type Abs on their surface.
•If Ags binds to IgE, the cell quickly releases enzymes to the environment -> degranulation
•Specific way: histamine, heparin and hyaluronidase loosen the tissues so the defence material can enter the interstitium.
• Non-specific way: inflammation -> release materials that loosen tissues -> immune cells and Abs can come to the Ags.

Question 22

10. Eosinophil granulocytes

Answer 22

-Also called acinophils. Type of WBC.
•Diameter: 12-14 μm
•Nucleus: less segmented (usually bi-lobed)
•Granules: red, actually lysosomes containing many enzymes. Horses: light, big and often covering nucleus
•Enzymes: Myeloperoxidase, histaminase, acidic and alcalic phosphatase, nuclease, aril-sulphatase etc.
•Lifetime: 3-5 days, 3-8 hours in circulation
•Significant antiallergenic effect
•No phagocytosis, only pinocytosis. Stronger chemotactic sensitivity.

Question 23

10. Mononuclear phagocytes

Answer 23

• Diameter: 15-25 μm (biggest)
• Nucleus: big, non-segmented, bean-shaped
• Granules: azurophyl
• Enzymes: myeloperoxidase, acidic and alcalic phosphatase, proteases, superoxid dismutase
• Present antigen
• Secretory function: prod. EC enzymes (collagenase, elastase, angiotensin-converting enzyme), interleukin-1, interferons and prostaglandines.
• Macrophage system: RES (reticulo endothelial system), RHS (reticulo histiocytic system).
• Dividing capasity
• Lifetime: in circ. 10-40 hours, in tissues 2-3 months
• Small circulation reaction. Extravasal supply; tissue macrophages of significant importance.

Question 24

10. Thrombocytes

Answer 24

(platelets)
•Size: 1.5-3.5 μm (smallest blood cells)
•Birds: 8-10 μm, with nucleus and able to phagocyte
•Number: 2-8x1011 pc/l
•Lifetime: 5-10 days
•No nucleus
•Passive movement, can be found mainly in circ.
•Granules: delta-granules; dark, store Ca2+, serotonin and ADP, or alpha-granules; cont. coag. factors.
•Plasma membrane: form invaginations and canaliculus-system connected to the surface. Wide glycocalix. CAM (cell adhesion molecules).
•Internal tubular system: peroxidase and prostaglandins
•Lysosomes and peroxisomes
•Actin cortex and microtubular cortex

Question 25

11. Haemostasis (without coagulation cascade mechanism)

Answer 25

-Complex defense reaction that prevents blood loss during vessel injury (intrinsic/extrinsic).
1.Vascular reaction: occurs immidiately - automatic process
•Contraction after injury: direct depolarization of SM cells. Neural: reflex. Tissue vasoconstrictor factors.
•Temporary stage, lasts for 6-10 min.
•Mechanical defense and decr. perfusion so thrombocytes stick to injured surface easilyer.
2.Thrombocyte reaction: aggregation of platelets - plug formation. Preventing further blood loss.
•Primary reaction: platelets bind with their surface receptors to neg. charges os injured endothelial areas.
•Secondary reaction: activated by microtubule system and secrations.
o Many filopodia formed, which help platelets bind to each other and exposed collagen fibres formes white thrombus.
o In intima injury this inhibition is automatically turned off -> thrombocytes prod. own stimulating factors; TXA2, 5-HT (serotonin) and adenosine-diphosphates, that stimulate other thrombocytes to incr. aggregation and secretion of coagulation factors.
3.Coagulation - fibrinogen-fibrin transformation: activated together with the activation of platelets.
•Enzymesystem stored in plasma and partly inactive thrombocytes.

Question 26

11. Stopping blood coagulation

Answer 26

1. Anticoagulation: untouched endothelial cells bind thrombin. Thrombin activates a plasma protein, the protein-C with help of calcium. Active protein-C directly prevents activation of key enzymes of both intrinsic and extrinsic activation complex
2. Fibrinolysis: thrombin stimulates activation of an inactive plasma protease, plasminogen. Forming plasmin is one of the most effective proteolytic enzyme that dissolves the fibrin-net.
3. Thrombolysis: together with loosening of clot, phagocytosis starts from inside also decr. the size of thrombus. Incr. perfusion plays a role in the demolition of the thrombus, too.

Question 27

12. Haemostasis: coagulation cascade mechanism

Answer 27

-Central “cascade”:
•Together w. activation of platelets, it starts activation of the enzyme system
o Enzyme system: stored in plasma and thrombocytes in inactive forms
o Causes form of fibrin net from fibrinogen of plasma (coagulation)
• Coagulation: result of ext tissue injury or the repeated enzyme activation caused by int. injury.
•Tissue injury stim coagulation factor X: last, common enzyme-activating system
• Activating factor Xa stim prothrombin-thrombin transform in presence of an activation complex
•Thrombin can turn plasma fibrinogen to loose fribin-net
•At last, coag. factors XIII-XIIIa are activated by thrombin, and stabilizes the formed fibrine-net.
-Extrinsic Way of Coagulation: ext tissue injuries
•Two important factors:
1. Inactive factor VII in plasma
2. Tissue factor
-Together it activates the first member of the common pathway, factor X
o With help of Ca, tissue factor fixes the other members of the cascade to the site of coagulation.
-Intrinsic Way of Coagulation:
•Microinjuries occur constantly.
•In case the smooth surface of intima turns rough
•Three components register the roughness and stim the central cascade by exaggerating this effect:
o Plasma factor XII connects to the surface
o It binds a macromolecular protein (kininogen)
o Binds and activates the enzyme (kallikrein) that transforms the factor XII to active XIIa