Plasma proteins and enzymes. Presented by Dr. Mohammad Saadeh The requirements for the Clinical Chemistry Philadelphia University Faculty of pharmacy

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1 Plasma proteins and enzymes Presented by Dr. Mohammad Saadeh The requirements for the Clinical Chemistry Philadelphia University Faculty of pharmacy

2 Introduction Blood proteins, also termed plasma proteins or serum proteins, are proteins present in blood plasma. proteins of blood plasma are examined for diagnostic purpose. Some plasma proteins are enzymes. The measurement the enzymes provide a sensitive indicator for tissue damage. Plasma contains >300 different proteins Many pathological conditions affect level of plasma proteins Mostly synthesized in the liver Some are produced in other sites

3 Functions of plasma proteins figure 13.1 page 224 Blood coagulation (examples: thrombin, fibrinogen and plasmin) defence against infection (humoral immunity such as immunoglobulins) Maintain plasma oncotic pressure (Albumin) Protease inhibitors: α 1 -antitrypsin (inhibit elastase) Enzymes: (examples: renin, coagulant factors, complement proteins removes cellular antigens) Buffering: plasma proteins help to maintain acid-base balance. transport of hormones drugs nutrients metabolites metabolic waste Examples: thyroxin binding globulin thyroxin Lipoproteins (cholesterol, triglyceride) Transferrin (iron)

4 Plasma proteins concentration g l; (<300 proteins) of this g/l is albumin g/l are serum globulins (transport protein, reactants of acut phase, globulins) biosynthesis: liver (most), lymphocytes (immunoglobulins), enterocytes (eg apoprotein B-48) degradation: hepatocytes, mononuclear phagocytic system (complexes of antigen-antibody, hemoglobin-haptoglobin)

5 Plasma proteins

6 Plasma proteins and enzymes Total proteins aid to the assessment of patients state of hydration: Increase total plasma proteins concentration due to a decrease in the volume of distribution (dehydration). Decrease total proteins results of an increase in plasma volume. Decrease total proteins of capillary increases because proteins will diffuse out into the interstitial space. Example; septicaemia or inflammatory. condition.

7 proteins Electrophoresis separate the plasma proteins into distinct bands: albumin, α1-globulins, α2-globulins, β-globulins and γ-globulins. (see Figure 13.3)

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9 Plasma proteins Transthyretin (prealbumin) Synthesis in liver. Transthyretin (prealbumin) (half life 2 days). Migrate before albumin in electrophoresis. Function: transport protein in serum and CSF that carry thyroxin and retinol-binding protein bound to retinol (vitamin A). Clinical significance: Prealbumin increased in: Patients receiving steroids Chronic renal failure Alcoholism Prealbumin decreased in: Poor protein nutrition or increase catabolism. Acute phase inflammatory response Hepatic damage

10 Plasma proteins Albumin (Alb) (normal value=40-60 g/l) Major plasma proteins Synthesis and secreted by liver. Biological half life in plasma about 20 days. Function: contributing to plasma oncotic pressure in both vascular and extravascular spaces. Albumin have ability to bind and transport a large number of compounds such as fatty acids, phospholipids, metal ion Ca+2, mg+2), amino acids, drugs, hormones (ex; cortisol) and bilirubin.

11 Plasma proteins Albumin (Alb); Clinical significance: Decrease serum level of albumin in (Hypoalbuminemia): Malnutrition Liver disease, cirrhosis (cause decrease synthesis of albumin) Renal disease (glomulars no longer functions nephrotic syndrome) because Increase excretion in urine Malignancy Ascites & oedema Burns haemorrhage and catabolic states Bind to drugs such as phenytoin Inherited such as Bisalbuminaemia: 2 bands for Alb in electrophoresis and the function change in Albumin. Analbuminemia: concentration of Alb less than 1 g/l, cause oedema. Increase serum level of albumin in (Hyperalbuminemia): Dehydration

12 Plasma proteins α1-antitrypsin or α1-globulins:( mg/dl) Major component around 90 % of serum fraction. Inhibitor of serine proteases enzyme such as elastase and trypsin. It is synthesized by hepatocytes and macrophages. Inherited disorder (decreased) of α1- antitrypsin can cause. Smoking Emphysema oxidizes a thiol group in methionine Cirrhosis at the active site of α1- antitrypsin result decreasing the activity Pizz homozygotes plasma α1- antitrypsin is reduced to 10%-15% Pizz heterozygotes plasma α1- antitrypsin concentration that are about 60% of normal.

13 Plasma proteins Haptoglobin (HP) or α2-globulins: ( mg/dl) Synthesized mostly by hepatocytes but also by other tissues such as skin, lung and kidney. Function: 1. preventing loss of iron through the kidneys and protecting the kidneys from damage by hemoglobin (suicide protein). HP is an acute phase protein Because Hb-HP complex is too large to pass through the glomerulus of kidneys. Hb-HP complexes are removed by macrophage system. Clinical significance: *increased plasma HP level in patients increase in any inflammatory process such as infection, burns, injury and allergy. HP increase in nephrotic syndrome. *Decreased plasma HP level in patients In Hemolytic anemia Decrease HP and increase reticulocyte indicate Spleen damage Liver damage drug-induce hemolysis. Decrease (Hp) without hemolytic anemia indicate liver damage or sever sepsis.

14 Plasma proteins α2-macroglobulin (α2m): High M.Wt protein (820 kda) Synthesis by hepatocytes and macrophages. Function: inhibitor of protease enzymes. Serum level of α2m increase in the nephrotic syndrome (characterized by large proteinuria). Why? It is too large to be filtered even though a damaged of glomerular basement membrane.

15 Plasma proteins Caeruloplasmin (α2-globulin) ( g/dl) It is copper carrying protein in the blood. Acute phase protein. Synthesized in liver. Caeruloplasmin functions as ferroxidase and superoxidase scavenger. Clinical significance: Elevated level of Caeruloplasmin in: Oestrogen-related effect pregnancy Low level of Caeruloplasmin in: Wilson disease Nephrotic syndrome

16 Transferrin (TF) (adults: mg/dl, Children: mg/dl) TF is a β globulin and TF is synthesize in the liver. TF are iron-transporting glycoproteins in the plasma that control the level of free iron in biological fluids TF is a serum protein that carries iron (2 mole of Fe +3 per mole of TF) through the blood stream to the bone marrow and other organs. TF binds to Fe +3 by two receptor and transported into the cell in a vesicle by receptor-mediated endocytosis. Normally, the iron bound to TF turns over times a day.

17 Clinical significance of Transferrin (TF) Transferrin iron-binding capacity (TIBC) is a medical laboratory test that measures the blood's capacity to bind iron with transferrin. NOTE: Transferrin level related to TIBC level. Transferrin and TIBC increased in : Iron deficiency anemia. During pregnancy. Transferrin and TIBC decreased in : Liver disease. Nephrosis Hemochromatosis. (Inherited disease cause accumulate of iron in tissue)

18 Ferritin Men : ng/ml; Women: ng/ml Ferritin serves to store iron in a non-toxic form and to transport it to areas where it is required. Clinical significance of Ferritin: *An increased plasma Ferritin level in patients iron overload disorders, such as hemochromatosis. Leukemia *Decreased plasma Ferritin level in patients iron deficiency anemia

19 Acute phase proteins and the acute phase response Acute-phase proteins are a class of proteins whose plasma concentration increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation or other related condition. This response is called the acute-phase reaction (also called acute-phase response). Clinical significance of acute-phase proteins: Positive acute-phase proteins (increase); increase synthesis by interlokin-6 C-reactive protein Fibrinogen, prothrombin Alpha 2-macroglobulin Ferritin Ceruloplasmin Haptoglobin Alpha 1-antitrypsin Negative acute-phase proteins (decrease) albumin, transferrin, retinol-binding protein, antithrombin

20 C-reactive protein (CRP) (<8mg/dL) Named because reacted with the somatic 'C' polysaccharide antigen of Pneumococcus. Inflammation release of interleukin-6 and other cytokines that trigger the synthesis of CRP by the liver. CRP binds to phosphocholine on the surface of dead and some bacteria. This activates the complement system (C1q), promoting phagocytosis by macrophages, which clears necrotic, apoptotic cell and bacteria. half-life of 18 hours. CRP rises within 6 hours of the onset of inflammation and peaks at 48 hours before beginning to fall.

21 C-reactive protein (CRP) Clinical significance of CRP: CRP increased in: Viral and bacterial infections Myocardial infarction (MI) or cardiovascular disease. rheumatoid arthritis Note: can be used to screen for inflammation. Note: Interferon alpha inhibits CRP production from liver cells which may explain the relatively low levels of CRP found during viral infections compared to bacterial infections.

22 Fibrinogen (1.5-3 g/l) Fibrinogen (Factor I) is a glycoprotein in vertebrates that helps in the formation of blood clots. Synthesis in the liver. fibrinogen molecule is a soluble, large, and complex 340 kda. Fibrinogen converted by thrombin into fibrin gel during blood clot formation. fibrinogen use in the investigation of some bleeding disorders. Increase fibrinogen levels include: Acute or chronic inflammatory Nephrotic syndrome Liver disease and cirrhosis Pregnancy or estrogen therapy Compensated intravascular coagulation decreased fibrinogen levels include: disseminated intravascular coagulation [DIC]). advanced liver disease

23 Other plasma proteins Proteins of Complement system investigate immunological disease and high levels are found in chronic infections. β 2 microglobulin is a component of MHC class I molecules, which are present on all nucleated cells can be elevated in multiple myeloma and lymphoma.

24 Immunoglobulins (Ig) An antibody (Ab), also known as an immunoglobulin (Ig). Ig behave mainly as γ-globulins. Ig play a key role in the defense mechanisms of the body. Types of heavy chain : α (alpha), γ (gamma), δ (delta), ε (epsilon), and μ (mu). This gives 5 types of immunoglobulins IgA, IgG, IgD, IgE, and IgM. Two type of light chain (κ,λ)

25 Immunoglobulins (Ig) Structure of immunoglobulins

26 characteristics of the Immunoglobulins

27 Immunoglobulins (Ig) Increase in concentration of Ig (polyclonal antibodies) in: acute and chronic infection autoimmune disease such as rheumatoid disease, Systemic lupus erythematosus. chronic liver disease.

28 Immunoglobulins (Ig) Paraproteins A paraproteins is an Ig produced by a single clone of cells of the B-lymphocyte ( monoclonal gammaglobulin). Decrease or normal concentration of Ig in patients with paraproteinaemia due to myeloma. (note: paraproteinaemia is excessive amounts of paraprotein) Immunoglobulins light chain found in urine known as bence Jones Protein and present in 75% of myeloma cases. paraproteins increase in 3% in people over the age of 70.

29 Immunoglobulins (Ig) Typical multiple myeloma Serum: Paraproteinaemia Normal Immunoglobulins Renal impairment Urea Creatinine Increase β 2 microglobulin is a component of MHC class I Increase Calcium Increase Urate Normal Alkaline phosphatase urine: bence Jones protein Anemia Serum: Typical benign Paraproteinaemia <30 g/l Increase Immunoglobulins Normal Calcium Normal Alkaline phosphatase Urine: NORMAL bence Jones protein NO anemia

30 Cytokines Cytokines are large group of autocrine (secrete by cell=chemical messenger) and paracrine (cell-cell communication) regulatory peptides, which modulate the activity of the immune system and are involved in the coordination of acute inflammation and the immune response. Four major of cytokines are recognized: Interleukins (IL), which are regulators of inflammation. Interferones (IF), antiviral agents, and inhibitory effect on cell growth. Colony-stimulating factors (CF), stimulate the growth of macrophages and white blood cells. Tumor necrosis factors (TNF), stimulate the proliferation of many cells, including cytolytic T-cells.

31 Enzymes Clinical Diagnosis Plasma enzymes can be classified into two major groups. First, enzymes are secreted into the blood.. Example, enzymes involved in blood coagulation. Second, enzyme are released from cells during normal cell turnover. These enzymes almost always function intracellularly, and have no physiologic use in the plasma. Increased plasma levels of these enzyme may indicate tissue damage so can be use for diagnostics and prognosis for the patient. (Figure 5.20).

32 Disadvantages of enzyme assays 1. Lack of specificity for a particular tissue or cell type. 2. Many enzymes are common to more than one tissue. This problem obviated to some extent in two ways: 1. Different tissues may contain two or more enzymes in different proportions. 2. Some enzymes exist in different forms (isoenzymes). Individual isoforms related to particular tissue.

33 Alkaline phosphatase (ALP) Responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. ALP ALP is the common name for a group of enzymes (5 isoenzymes) ALP is produced and present in high concentration in liver, bone, placenta and intestinal etc. Every tissue produce one characteristic form so, electrophoresis is used to determine the type of ALP to know the origin tissue. skeletal).

34 Clinical significance: diagnosis of two groups of conditions; ALP increase in; 1. hepatobiliary disease Alkaline phosphatase (ALP) (obstructive jaundice, cirrhosis, viral hepatitis and metastatic). 2. bone disease Osteomyelitis Paget's disease Primary hyperparathyroidism with bone involvement. ALP Physiologically increase in Pregnancy Childhood Fatty meals (intestinal ALP). To determine the damage tissue with high ALP (vvi) ALP and γ-glutamyl transferase that is found in liver but not in bone indicate liver disease. Increase ALP with hypercalcemia (increase level of calcium in plasma) multiple myeloma or leukemia, bone disease, osteomyelities and Paget's disease.

35 Alkaline phosphatase (ALP) ALP Physiologically increase in Pregnancy Childhood Fatty meals (intestinal ALP).

36 Acid Phosphatase (ACP) ACP is present in prostate, liver, bone, spleen, kidney, erythrocyte and platelets. Orthophosphoric monoester + H 2 O alcohol + H 3 PO 4 (remove phosphate group) Clinical significance: Elevated levels of serum Acid Phosphatase (ACP) in: Confirming and evaluating a diagnosis of prostatic carcinoma. (tumor marker). Paget s disease, hyperparathyroidism with skeletal involvement. Cancers which have invaded the bones.

37 Aminotrasferases Two used in diagnosis; aspartate aminotrasferases (AST) alanine aminotrasferases (ALT). 1. aspartate aminotrasferases (AST) or glutamic-oxaloacetic transaminase (GOT). vvi Functions: The enzyme catalyses the transfer of amino groups during the metabolism of Amino acids and, alpha-ketoacids. Tissue source high level of AST in cardiac, liver & SK muscle. low level of AST decrease in kidney, pancreas &erythrocyte. Clinical significance: AST levels are elevated in: 1. myocardial infarction (MI) 2. liver disease (hepatocellular damage). heart attack (myocardial infarction (MI)) primary muscle disease recent surgery and severe burns

38 Aminotrasferases Clinical significance: AST or (GOT) levels are elevated in: Maximum elevations (> 20 times normal) *Acute viral hepatitis Sever tissue hypoxaemia High level (10-20 times normal) *myocardial infarction (MI) High level (5-10 times normal) *Chronic hepatites vvi vvi Cholestasis (bile cannot flow from the liver to the duodenum) High level (2-5 times normal) Metastatic hepatic tumors Acute pancreatitis Hemolytic anemia Hemolysis (ex; statins)

39 Aminotrasferases AST (GOT) increased 4-8 hours following a myocardial infarction (MI), reaching its peak in 2-3 days and declining on the fifth and sixth days. AST is not a specific or sensitive enough marker for the diagnosis of mycocardial infraction so cardiac troponins is much used.

40 Aminotrasferases 2. alanine aminotrasferases (ALT) or Glutamic-Pyruvic Transaminase (GPT). Functions: ALT catalyzes the transfer of the amino group from L-alanine to α- ketoglutarate resulting in the formation of pyruvate and L-glutamate. Tissue source high level in liver low level in cardiac, kidney & skeletal muscle. ALT is considered more liver-specific than AST.

41 Aminotrasferases Clinical significance: ALT levels are elevated in: Acute or chronic hepatitis (cellular damage) cirrhosis or scarring of the liver with loss of function. Viral hepatitis. cholestasis or congestion of the bile ducts metastatic carcinoma. Extensive liver damage from toxins or drugs. ALT is considered more liver-specific than AST. vvi ALP used to detect and evaluate treatment of acute hepatitis disease. ALP distinguish between MI and hepatic damage (used with AST). ALP used to assess the hepatotoxicity of some drugs.

42 γ-glutamyl transferase (GGT) GGT present in high concentration in liver, kidny and pancreas. Sensitive for hepatobiliary disease. Function: GGT catalyzes the transfer of the γ-glutamyl group from Glutathione to amino. Clinical significance: GGT levels are elevated in: High level (>10 times normal) Cholestasis alcoholic liver disease High level (5-10 times normal) Acute and chronic Hepatitis Cirrhosis (without cholestasis) Pancreatitis High level (< 5 times normal) Excessive alcohol ingestion Enzyme-inducing drugs Congestive cardiac failure

43 γ-glutamyl transferase (GGT) GGT elevated in patients with liver diseases taking alcohol, phenytoin, pheonobarbital and rifampicin and can remain elevated for up to 3-4 weeks. increase both ALP and GGT indicate liver disease specially cholestasis. VVI Measuring isoenzyme of ALP and GGT that is found in liver but not in bone so, identify the origin tissue of ALP. VVI

44 Lactate dehydrogenase (LDH or LD) LDH is an enzyme found in nearly all living cells. LDH level are high in liver, skeletal muscle, kidney and erythrocytes. LDH exists as tetrameric composed of H and M subunits that form the five isoenzymes, LDH-1 (4H), LDH-2 (3H1M), LDH-4 (1H3M), LDH-5 (4M). Function: Lactate dehydrogenase catalyzes the oxidation of lactate to pyruvate with simultaneous reduction of NAD to NADH. LDH isoenzyme distinguished by electrophoresis due to different mobility. Anode (+) Cathode (-) H H H H H H H M H H M M H M M M M M M M LDH 1 (H 4 ) LDH 2 (H 3 M) LDH 3 (H 2 M 2 ) LDH 4 (HM 3 ) LDH 5 (M 4 )

45 Lactate dehydrogenase (LDH or LD) LDH-1 and the substrate α-hydroxybutyrate dehydrogenase (HBD) increase in: Increased levels of LDH-1 and LDH-2, are associated with myocardial infarction (MI); rise hours, Peak 48 hours; remain ten days. Hemolytic crises in sickle cell anemia. LD3 in the lung. Increased levels of LD4 and LDH-5 in liver disease and skeletal muscle.

46 Creatine kinase (CK) Creatine Kinase exists as dimeric molecules composed of M and B subunits that form the isoenzymes CK-MM, CK-MB, and CK-BB. CK-MM are distributed primarily in the skeletal muscle. CK-MB are distributed primarily in the heart muscle. CK-BB is present mainly in the brain and in tissues composed of smooth muscle. Functions: storage of energy in the form of phosphocreatine. Creatine Phosphocreatine Clinical significance: CK-MB increased in MI and rarely skeletal muscle damage. CK-MB detection is of importance in determining the degree of the injury and the efficacy of the treatment. CK and LDH isoenzymes provides a definitive diagnosis of acute myocardial infarction (MI). Note: CK begins rise within 4 8 hours following onset of chest pain, reaches a peak of activity at 24 hours, and returns to baseline after hours

47 Amylase Amylase is found in the salivary glands and exocrine pancreas. Functions: that catalyses the hydrolysis of starch into sugars (act on α-1,4- glycosidic bonds). Clinical significance: In acute pancreatitis α-amylase starts to rise approximately 4 hours after the onset of pain, reaches a peak at 24 hours and remains elevated for 3-7 days. Amylase increase in acute abdominal disorders, salivary gland disorders, and macroamylasamia.

48 Lipase lipase catalyzes the hydrolysis of lipids to alcohol and fatty acids (RCOOH). Clinical significance: Increase Pancreatic lipase is important for diagnosis of pancreatic diseases and for associated monitoring of therapeutic effects (more specific).

49 Cholinesterase (CHE) CHE enzyme secreted by the liver into the bloodstream. Function: cholinesterase break down an acetylcholine by preventing the accumulation of acetylcholine and the overstimulation of muscles and nerves. symptoms of overstimulation of muscle and nerve fibers cause difficulty in breathing or death. Clinical significance: Low plasma activity of CHE in: Physiologically during pregnancy chronic hepatic dysfunction. Liver disease Organophosphate (pesticides ) poisoning so *Cholinesterase test helps doctors determine whether or not an individual is poisoned Cholinesterase hydrolysis a muscle-relaxant drug, used in anaesthesia (suxamethonium). Cholinesterase must be examined to avoid anesthesia in abnormal cases of cholinesterase activity. vvi

50 Tumor Markers Alpha-feto protein (AFP) Tumor markers are substances, usually proteins, that are produced by the body in response to cancer growth. AFP is a major plasma protein produced by the yolk sac and the liver during fetal development. AFP levels (>500ng/ml) are increased in >90% of patients with hepatocellular cancer. AFP are useful in monitoring the response to therapy of hepatocellular cancer. AFP levels increase in pregnancy (false positive). vvi

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