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Blood Biochemistry

Blood Biochemistry. Tissue Chemistry & Biological Fluids. Biochemistry has passed from a state of descriptive to quantifiable science. As a biochemist, you should always be interested in things about metabolic sequences:

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Blood Biochemistry

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  1. Blood Biochemistry

  2. Tissue Chemistry & Biological Fluids Biochemistry has passed from a state of descriptive to quantifiable science. As a biochemist, you should always be interested in things about metabolic sequences: • The description of the enzymes & chemical changes that comprise the metabolic sequence • The rate at which material can be transformed by the sequence • The amount of material utilized by the sequence among living things • The nature of the control mechanisms which adjust the amounts of material utilized by the sequence

  3. Roughly, the contributions of the different tissues to the body's metabolism are proportional to the weights of the tissue and the biological fluids

  4. From the following table… • It is not the sheer mass of tissue which determines its quantitative contribution to metabolic activity • Activity of tissue is determined by its enzyme content

  5. The body can be crudely divided into two components:

  6. Vertebrates have evolved 2 principal mechanisms for supplying their cells with a continuous & adequate flow of oxygen: • A circulating system that actively delivers oxygen to the cells • Acquisition of oxygen • The oxygen carriers in vertebrates are the proteins hemoglobin & myoglobin

  7. Blood • In a normal weight, there is about 5-6 liters of blood (12%) or 85ml/kg • It circulates as a homogenous suspension of erythrocytes, leukocytes & platelets in a solution of proteins, inorganic ions, & low-molecular weight organic compounds

  8. Functions of the Blood • Transport of nutrients • Exchange of respiratory gases • Transport of waste products • Distribution of hormones & other regulatory substances • Protection against microorganisms • Acid-base, electrolyte & water homeostasis • Heat regulation • Prevention of excessive hemorrhage by coagulation

  9. General Composition • By volume, 40-45% of the blood consists of erythrocytes, leukocytes & platelets • 1 mm3 of blood contains: 5 x 106 RBCs; 5-103 WBCs; 5-105 platelets

  10. The Packed Cell Volume (PCV Hematocrit) PCV Hematocrit = Volume of Red Cells/ Volume of whole blood x 100 • Expressed as volume of erythrocyte per liter of whole blood • Normal adult males = 41-53; adult females = 36-46 • Hematocrit used to determine PCV • Color of supernatant plasma gives rough idea of bilirubin content & is often a useful clue about the nature of anemia: • White plasma ----- iron deficiency anemia • Lemon yellow plasma ----- Hemolytic or Megaloblastic anemia

  11. Blood Volume & the Hematocrit • Rarely necessary to have an accurate blood volume • Hematocrit (HCT) is the volume percentage of erythrocytes in whole blood • HCT is obtained by centrifugation • The specific gravity of WBC's intermediate between plasma & RBC, thus forming "Buffy Coat"

  12. Errors in the Estimation of HCT • Usually up to 5% of the apparent RBC mass is plasma • HCT differs according to blood source: - Some particles when centrifuged tend to accumulate in the center of the tube - HCT value is affected by movements of fluid (hydrostatic pressure)

  13. Clinical value of HCT • HCT is important in the diagnosis of anemia • Rough estimation of blood loss after hemorrhage

  14. Continuation… Whole Blood • Whole blood – formed elements = plasma • Plasma – Clotting factors = Serum

  15. Physical Characteristics • Arterial blood is crimson • Venous blood is darker red • Specific gravity = 1.035-1.090 & the viscosity is 5-6 times that of water • Specific gravity of plasma = 1.015-1.035 • Ph = 7.3-7.5

  16. Erythrocyte Sedimentation Rate • Rate of settling of RBCs after blood is drawn • In healthy men : 1-3mm/hr; 4-7 mm/hr in young women • Low ESR in patients with anemia • Follows Stoke's Law (settling velocity) with an equation Where: Vs is the particles' settling velocity (m/s) (vertically downwards if ρp > ρf, upwards if ρp < ρf), r is the Stokes radius of the particle (m), g is the standard gravity (m/s2), ρp is the density of the particles (kg/m3), ρf is the density of the fluid (kg/m3), and η is the fluid viscosity (Pa s).

  17. Continuation… • ESR greatly increased during menstruation & normal pregnancy • Increased rate also found in septicemia & pulmonary tuberculosis (increased globulin & fibrinogen content of plasma; also in elevated cholesterol & phospholipid levels • Inflammation of various types that cause cell necrosis will cause rate of RBC to fall, but the viscosity remains unchanged.

  18. Continuation… In alcoholic cirrhosis there is a rise in plasma bile acids & membrane cholesterol levels may rise by 55%. This has 2 effects: • Cholesterol to phospholipid ratio is increased, reducing membrane flexibility • Increased cholesterol content also raises total lipid present, expanding its surface area • Increase by 8% in the total lipid is enough to cause formation of spherocytes (removed from the circulation as a result of alteration in size, shape and flexibility) • Elevated levels of plasma bile acids (mainly cholic & deoxycholic acids) are observed in obstructive jaundicewith similar consequences for the RBC membrane

  19. Plasma • Straw colored fluid with specific gravity from 1.015-1.035 • Specific gravity of plasma is related to its protein content • Contains 90-92% water

  20. Continuation… Blood owes much of its physiological importance to high water content: • Maintaining blood pressure • Important for heart regulation& in osmotic exchange between body fluid & compartments

  21. Plasma Composition • The solutes of the blood plasma constitute ≈ 10% of the volume • Protein ≈ 7% • Inorganic salts ≈ 0.9% • Other organic compounds ≈ the rest other than proteins.

  22. Separation of plasma proteins Based on the different mobility in an electric field • Electrophoresis – widely used • Isoelectric focusing • Immunoelectrophoresis – separates proteins on the basis of electrophoretic as well as immunologic properties Albumin Beta Alpha 2 Alpha 1 Gamma

  23. Albumin & Globulins Albumin & Globulins • Comprise most of the proteins in the blood plasma • Colloidal osmotic pressure (from the proteins of the plasma) is the force that opposes the hydrostatic pressure in the capillaries • Better terminology should be "potential osmotic pressure" or "osmotic tendency"

  24. Proteins move in electric field by the charge they carry… Major fractions include: • Albumin (54-58%) • α1 globulins (6-7%) • α2 globulins (8-9%) • β1- globulins (13-14%) • Gamma globulins (11-12%)

  25. Enzymes of Plasma • Most plasma enzymes do not have metabolic roles in plasma with the exception of those involved in coagulation • Activity of certain plasma enzymes is useful as index of certain abnormal conditions: - Serum amylase – elevated in acute pancreatitis - Acid phosphatase – in cases of prostatic cancer - Alkaline phosphatase – in hepatic obstruction and bone diseases

  26. Assay of tissue enzymes in plasma • When organs are damaged part of their enzyme complement in released into the plasma. • In a healthy persons, levels of intracellular enzymes are very low & a result of cellular turnover • Tissues contain 103-104 times higher content of soluble enzymes within their cells • Intracellular enzymes released into the plasma are inactivated & removed within days • Amount of enzyme released depends on the concentration of that enzyme & extent of tissue damage • Knowledge of cellular location of enzyme provides good clinical information • In practice, enzyme assays are most useful in detecting damage to the liver, muscles and blood cells

  27. Some enzymes commonly assayed as part of clinical diagnosis

  28. Continuation… • CPK (Creatinine phosphokinase) & LDH1 (Isoenzyme of LDH) indicate amounts of myocardial infarct. If no further damage occurs, levels return to normal. • Congested liver can be due to inefficient pumping of the right side of the heart. • Most patients with metastatic prostatic carcinoma have elevated plasma phosphatase levels. RIA is used for the detection of this enzyme. Enzyme assays may also reveal other organ involvement…

  29. Erythrocytes Circulating erythrocytes are derived from erythropoietic cells (or erythron), the precursors of erythrocytes. RBCs arise from mesenchymal cells present in bone marrow Major functions • Transport of oxygen from the lungs to the tissues • Controls blood pH (CO2)is converted to bicarbonate by carbonic anhydrase = major buffering system) • RBCs lack nucleus & other organelles; utilizes anaerobic metabolism

  30. Structure & Composition • RBC s have a biconcave disc shape(6-9µm in diameter; 1 µm thick; 2-2.25 µm at the periphery) • Most of the solid matter is hemoglobin ( the conjugated protein responsible for the red color of the blood) • Behaves like an osmometer

  31. The Erythrocyte Membrane • Composed largely of protein (49%) & lipid (43%) with a small amount of carbohydrate (8%) • Has a cytoskeleton which controls the shape of the membrane & limits the lateral mobility of some intrinsic proteins • Some of the protein is glycoprotein covalently linked to CHO (Sialic acid)

  32. Membrane Changes in Diseases Mature RBCs synthesize very little lipids but: Sphingomyelin Phosphatidylcholine in the outer half of the bilayer those in plasma lipoproteins Cholesterol exhanges freely with serum cholesterol The important factor that affects this exchange is the activity of the plasma enzyme Lecithin-Choelsterol Acyl Transferase (LCAT) – responsible for the formation of majority of esterified cholesterol and is inhibited by bile acids

  33. Erythropoiesis During gestation, erythrocytes are formed in varioustissues occurring successively in: • Yolk sac– main site for the 1st weeks of gestation • Liver & Spleen- from 6 weeks to 6-7 months & can continue to produce until about 2 weeks after birth • Lymph Nodes

  34. Continuation… From 6-7 months of fetal life onwards… the bone marrow is the only source of new blood cells

  35. Continuation… • Erythroid cells in the bone marrow are called normoblast (a large cell with dark blue cytoplasm, a central nucleus with nucleoli & slightly clumped chromatin Reticulocytes • A reticulocyte stage results when the nucleus is finally extruded from the late normoblast. In this stage it still contains some ribosomal RNA and can still synthesize Hb • Reticulocytes spends 1-2 days each in the circulation & bone marrow before it matures mainly in the spleen when RNA is completely lost • A single pronormoblast usually gives rise to 16 mature red cells

  36. Substances needed for erythropoiesis The bone marrow requires many precursors to synthesize new cells: • Metals: Iron, manganese, cobalt • Vitamins: B12, folate, ascorbic acid • Amino acids • Hormones

  37. Hemolysis • Maybe produced by substances that dissolve or change the state of membrane lipids (ether, chloroform, bile salts & soaps) . • Certain biological toxins (venomous snakes & hemolytic bacteria) • Physical forces (UV rays, freezing, thawing) • Aging – this is why whole citrated blood cannot be used after 5-7 days

  38. Red Cell Metabolism The components required for these include: • ATP – maintenance of membrane function • 2,3 –diphosphoglycerate (2,3 – DPG) to modulate O2 affinity • NADPH – to prevent hemoglobin denaturation • NADH – to maintain the heme in the Fe(II) state

  39. Continuation… • The predominant metabolic fuel is glucose where they serve as gluconeogenic precursors • The 2 ATP molecules are utilized in the ion pump in the cell membrane • Failure to produce enough ATP results in an ability to maintain ionic balance leading to accumulation of Ca2+ and shape change

  40. Continuation… • 2,3 DPG is a metabolite unique to the RBC. At a concentration of 4-5mM, it is almost equimolar to Hb • 20-25% of 1, 3 DPG pass to 2, 3 DPG by mutase, therefore ATP yield decreases from glucose • 2,3 DPG depends on the relative rates of the mutase& phosphatase reactions

  41. Glutathione • Can be used for the removal of H202. • This reaction protects the membrane from oxidative damage. • A deficiency of any enzyme of the glycolytic, phosphogluconate or GSH-GSSG pathway may seriously compromise the energy dependent maintenance of membrane integrity.

  42. Hereditary Hemolytic Anemias Have been associated with deficiencies of the following enzymes: • Enolase enzyme – deficiency leads to decreased ATP required to maintain the biconcave shape of RBC • Glucose-6-P-Dehydrogenase – deficiency may result in increased hemolysis & severe hemolytic anemia • Pyruvate Kinase - deficiency may lead to bizarre modelwhich is extremely fragile and readily hemolyzed • Other enzymes such as hexokinase,glucose –phosphoisomerase, phosphofructokinase, triose phosphate isomerase, 2-3 diphosphoglycerate dismutase

  43. Erythrocyte Destruction • Senescent erythrocytes are engulfed primarily in the reticuloendothelial cells of the spleen • Free hemoglobin is released and binds to plasma proteins (e.g. haptoglobin) • Complex is transported to liver where Hb portion is split • Heme portion is transported to plasma& converted to bilirubin; excreted in the bile • Iron is released & stored in the liver for reuse

  44. Hemoglobin • 1 liter of blood usually contains 150gof hemoglobin; each gram can combine with 1.34ml of oxygen

  45. Continuation… • 1 liter of blood can carry 200ml of oxygen, 87 times higher than plasma alone. • Each RBC contains ≈ 640 million Hb molecules

  46. Hemoglobin Structure • The 4 chains are held together by non-covalent bonds • There are 4 binding sites for oxygen • The Hb molecule is nearly spherical; packed together in a tetrahedrical way

  47. Continuation… • The amino acid sequence of hemoglobin is known for 20 species. However there are 9 positions in the sequence that contain the same amino acid in nearly or all species studied.These conserved positionsare especially important for the function of hemoglobin: • Some of them are involved in oxygen binding sites • Stabilizing the molecule via forming H-bond between the helix • Some (e.g. GLY) for easy contact between the chains • Some (e.g. PRO) to terminate the elix • The non-polar residues (Alanine, Isoleucine) are important because reversible oxygenation of heme group depends on its location where it is protected from water.

  48. Continuation… • Normal hemoglobin is of several types containing 4 sub-units made up of various combinations of 4-5 different related peptide chains

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