CA 2 Biochem PDF

Clinical Enzymology 1. Active enzyme - Secreted into the plasma by certain organ - Presence in small quantities - Granule-zymogen storage vesicle - All digestive enzyme were produced and kept as a zymogen-inactive proenzyme 2. Enzyme with no physiologica...

Clinical Enzymology 1. Active enzyme - Secreted into the plasma by certain organ - Presence in small quantities - Granule-zymogen storage vesicle - All digestive enzyme were produced and kept as a zymogen-inactive proenzyme 2. Enzyme with no physiological function in the plasma - Mostly intracellular - Steady state in healthy individual - Any changes to the enzyme levels in plasma indicate diseased situation Factors influencing plasma enzyme assay 1. Analytical - [substrate & product], pH, temperature, buffer, activator/inhibitor 2. Physiological - Age, gender, physiological condition Isoenzyme: catalyzed the same reaction but migrated differently in electrophoresis Intracellular location Enzymes Cytoplasm LD, ALT, 30% AST Mitochondria 70% AST Golgi complex, ER CHS, AMS Lysosome ACP Membrane GMT, ALP Enzymes of Clinical Significance Enzymes Source of blood elevation ALT hepatopathy AST MI, hepatopathy GMT Hepatopathy (alcohol, drugs) ALP Biliary tract disease, bone disease ACP Prostatic cancer  CK MI (CK-MB), muscle diseases AMS pancreatitis LPS pancreatitis CHS Hepatopathy (alcohol, drugs) - decreased Functional enzymes - Produced in the liver or other tissue and function in the plasma - Substrate in the plasma - Determination of enzymes were not used for diagnosis of a disease Non-functional enzymes - Secreted into the plasma due to disruption of the cell - During normal condition, its activity is low - Excreted out from blood by excretory organ and degradation metabolism - Increase in activity in the plasma due to: damage to the cell membrane, increased synthesis, changes in the permeability of cell membrane. - Used as an indicator for diagnosis of certain diseases. - Level of the enzyme activities is always correlated with the degree of tissue damage 1. Lactate Dehydrogenase (LDH) 2. Aspartate Transaminase (AST) 3. Alkaline Phosphatase (ALP) 4. Alanine Aminotransferase (ALT) 5. Creatine Kinase (CK) Aspartate Transaminase (AST) // Serum glutamate oxaloacetate transaminase (SGOT) Catalyse the reaction of: L-Asp + a-ketoglutarate > Oxaloacetate + L-glutamate - Presence in high concentrations in cardiovascular cell, skeletal muscle, liver, kidney, and erythrocytes - Damage to all the above tissues will increase the levels of AST in the plasma (hemolysis, MI, acute/toxic hepatitis, cirrhosis, jaundice, trauma or surgery) Alanine Transaminase (ALT) // Serum glutamic pyruvic transaminase (SGPT) Catalyse the reaction of: L-Ala + a-ketoglutarate > Pyruvate + L-glutamate - Presence in high concentration in liver, skeletal muscle, kidney and heart. - Circulatory failure and shock will increase its level - Acute hepatitis or intoxication - Cirrhosis, liver congestion due to cardiac congestive condition - Surgery or extensive traumatic During virus hepatitis infection, the increase of AST & ALT level in serum will be detected early before the increase in bilirubin. Lactate Dehydrogenase (LDH) Catalyse the reversible reaction of lactate and pyruvate with the presence of NAD+/NADH - Easy to assay - Increase in concentration in cardiac cell, skeletal muscle, liver, kidney, brain and erythrocytes - Abundant in the body, therefore not a good diagnostic tools - Secreted due to haemolysis or hematology related conditions - Anemia, acute leukemia & lymphoma, MI, rejection of kidney transplantation, viral hepatitis Creatine Kinase (CK) / Phosphokinase - Presence in abundant at cardiac muscle, skeletal muscle, and in brain and smooth muscle - Secreted due to hemolysis - During neonate - MI, muscle dystrophy, hypothyroidism CK Isoenzyme Have 2 subunit, M & B 1) CK-MM isoenzymes are abundant in skeletal muscle and cardiac muscle. Can be detected in normal people. 2) CK-MB represents 35% of CK activity in cardiac muscle & maltose - Increase in pancreatic juice and saliva - Also in gonad tissue, fallopian tube, skeletal muscle, and adipose tissue - Due to decrease in molecular weight, it is secreted into the urine - Acute pancreatitis diagnose: [concentration] increase & abdominal pain Pancreatitis Occur due to rise in digestive enzymes in the plasma such as amylase, lipase and trypsin. - Damage to the pancreas and its ductus causes amylase to enter blood circulation and be excreted out of the body through the urine. Alkaline Phosphatase (ALP) - Organic phosphate hydrolysis to pH increase - Presence in most tissue - Important in bone classification - [Increase] osteoblasts, hepatobiliary tract cells, intestinal wall, renal wall & placenta - Isoenzyme increase in liver, not in damaged hepatocytes but blockage of bile ducts (cholestasis) Acid Phosphatase (ACP) - In prostate cells, liver, erythrocytes, platelets and bones - To diagnose carcinoma prostate by measuring plasma prostate specific antigen (PSA) - ACP flow from the prostate through prostatic ducts to urethra & a little can be detected in the plasma Y-glutamyl-transferase (GGT) - Abundant in hepatocytes, kidney, pancreas and prostate - GGT plasma activity is high in men - Drugs/alcohol can catalyze the synthesis of enzyme without damaging the cells - Secreted into the plasma from the hepatic cells that suffer hepatobiliary - y-GT increased in liver disease especially in obstructive jaundice - y-GT levels are used as a marker of alcohol induced liver disease and liver cirrhosis Examples of diagnosed diseases with enzymology techniques 1) Pulmonary embolism: non-functional enzymes (LDH, CPK, B-hydroxybutyrate dehydrogenase) 2) Pulmonary infarct: LDH-3, Alkaline phosphatase 3) MI: a. AST (enzyme leakage from necrosis myocardial muscle cells. AST > 1500 IU/L poor prognosis. b. LDH (increase within 12-18 hours following acute MI, reach the peak in 2-3 days & back to normal in 7-10 days. Isoenzyme LDH is H4 &MH3. c. B-hydroxybutyrate dehydrogenase (reach the peak for 12 weeks following MI). d. CPK (increase in activity in 4 hours following MI, reaching the peak within 24-36 hours) 4) Liver disease: Alkaline phosphatase, leucine aminopeptidase, 5’nucleotidase, AST and ALT, LDH5 isoenzyme, glutamic dehydrogenase. 5) Tay-Sachs, Psychosis, Meningitis: AST, CK, LDH2 & LDH3 isoenzyme 6) Cancer: LDH, acid phosphatase, amylase, lipase, aldolase, B-glucuronidase 7) Lung carcinoma: LDH 1 2 3 8) Prostate carcinoma: Acid phosphatase 9) Kidney disease - Enzymes in plasma: LDH, Alkaline phosphatase, AST - Enzymes in urine: Muranidase, catalase, nitrate reductase, sulfase, urokinase, B-glucuronidase 10) Skeletal muscle disease: LDH5, ALT, CPK-3 Metabolism disorder Catabolism: Process of macromolecule breakdown in cells. The intermediates will be formed. - Carbohydrates: glycolysis, HMP shunt, Krebs cycle, fructose, galactose metabolism - Protein: proteolysis and amino acid catabolism - Lipid: lipolysis, beta-oxidation and ketogenesis Anabolism: Synthesis of macromolecules from intermediate compounds. Requires energy. - Carbohydrates: gluconeogenesis, glycogenesis - Protein: protein & amino acid synthesis - Lipid: lipogenesis, cholesterogenesis, triglyceride & phospholipid synthesis Metabolism disorder can probably cause: 1. Accumulation of intermediates (acidosis, hypercholesterolemia, hyperammonemia) 2. Decrease or absence of final outcome of metabolism (abetalipoproteinemia, thalassemia, hemolytic anemia cause G6PD deficiency) 3. Increase in other metabolism (diabetes mellitus type I) Causes of metabolic disorders: Lack of certain enzymes in the metabolism Genetic factors can cause the lack of or absence of this enzyme. Often a rate-limiting enzyme. Example: Anemia caused by G6PD deficiency & hyperlipoproteinemia type I. Lack of hormones that regulate metabolism Genetic and nongenetic factors. Example: Diabetes Mellitus type I. Deficiency of cell membrane receptors for These receptors are usually involved in the metabolism role of hormone or regulators. Example: Diabetes Mellitus Type II Inhibition of certain enzymes caused by certain substances ingested or entering cells. Example: inhibition of cytochrome oxidase by carbon monoxide and lead. Lack of certain proteins Essential protein in metabolism. Ex: Wilson’s disease ; lack of ceruloplasmina to transport copper. Changes in protein primary structure Ex: sickle cell anemia; changes of one amino acid at the RBC globin beta chain caused RBC to become sickle shape and easily undergo hemolysis. Metabolism disorder of one substance can Ex: disorders in protein synthesis interfere interfere with the metabolism of another with the synthesis of enzyme molecules substance Deficiency of substrate or coenzyme reaction Ex: deficiency of several types of vitamin B complex can causes disease such as Pellagra or Beri-beri Increase in certain hormones may increase Ex: increased thyroid hormone metabolic rate (hyperthyroidism) Examples of metabolic disorders 1. Hyperlipoproteinemia type II Absence of LDL receptors due to genetic disorders in its synthesis - hereditary disease. The result of this disease is an increase in LDL or cholesterol in plasma, which can be related to arteriosclerosis. 2. Diabetes mellitus type I The absence of insulin causes metabolic disorders of glucose. This will increase the metabolism of fatty acids > ketogenesis that ultimately causes ketoacidosis. 3. Anemia caused by G6-PD deficiency The absence of glucose-6-phosphate dehydrogenase enzyme - no pentose phosphate pathway > no NADPH + H+ > RBC easily undergoes hemolysis by oxidative stress caused materials such as H2O2, nitrate and quinine. 4. Hyperammonemia type I Deficiency of carbamoyl phosphate synthetase I enzyme can cause hyperammonemia and death 5. Jaundice caused by inherited disease Results from absence of UDP-glucuronyl transferase in liver cells. Ex: Crigler-najjar & Gilbert syndrome. 6. Lipidosis Lack of enzymes in the metabolism of lipid/phospholipid, accumulation of lipid. Ex: Hand-Schuller-Christian disease, Niemann-pick disease, Tay-Sachs disease, Gaucher’s disease 7. Glycogen storage disease Deficiency of glycogenolysis enzyme, glycogen in the liver cannot be broken down and will accumulate. 8. Alkaptonuria Inherited metabolic disease; caused by lack of homogentisate 1,2-dioxygenase which plays an important part in tyrosine metabolism. Amino Acid inborn error of metabolism (IEM) - IEM usually autosomal recessive Diagnostic confirmation: Direct biochemical assays of metabolites/their metabolic by-products, DNA studies, or neuro-radiology Disease of the AA metabolism - Impair the synthesis and degradation of amino acid - IEM occurs from a group of rare genetic disorders; the body cannot metabolize food components normally. Caused by defects in the enzymes involved in the biochemical pathways that break down food components. - Most are due to a defect in an enzyme or transport protein, which block the metabolic pathways - Effects are due to toxic accumulations of substrates before the block. Amino acid transport disorders: Cystinuria, dicarboxylic aminoaciduria, Hartnup disease Amino acid storage disorder: Glutaric acidemia type II Inborn error of metabolism Urine odor Glutaric acidemia Sweaty feet Maple syrup urine disease Maple syrup HYpermethioninemia Boiled cabbage Phenylketonuria Mousy or musty Trimethylaminuria Rotten fish Alkaptonuria - Black urine disease is a rare genetic disorder of phenylalanine and tyrosine metabolism. - Due to defect in the enzyme homogentisate 1,2-dioxygenase, which participates in the degradation of tyrosine - As a result, homogentisic acid (alkapton) accumulates in the blood and excretes in urine in large amounts. - Excessive alkapton causes damage to cartilage (ochronosis, lead to osteoarthritis) and heart valves as well as precipitating kidney stones. - Ear wax exposed to air turns red or black after several hours. Diagnosis: Paper/TLC, plasma and urine can be used. Plasma level: 6.6 mg/ml, urine level: 3.12 mmol/mmol of creatinine. Phenylketonuria (PKU) A silent disorder, if untreated, could lead to brain damage and developmental disabilities. - Associated with behaviour disorders, cataracts, skin disorders, and movement disorders. - Clinical symptoms of PKU are caused by the accumulation of phenylalanine in the blood that is 30-50 times higher than normal. - Lead to production of phenylalanine metabolites such as phenylpyruvate, phenylacetate, and phenyllactate. Ornithine Transcarbamylase (Urea Cycle disorder) Type 2: acute metabolic crisis. Life-threatening in infancy - Develop vomiting, respiratory distress, lethargy, may slip into coma. Tyrosinemia Hereditary tyrosinemia is a genetic IEM associated with severe liver disease in infancy. Autosomal recessive. - Caused by an absence of the enzyme fumarylacetoacetate hydrolase (FAH) - Lead to an accumulation of toxic metabolic products in various body tissues, resulting in progressive damage to liver and kidneys. Acute Tyrosinemia - Babies may show poor weight gain, an enlarged liver and spleen, a distended abdomen, swelling of the legs, and an increased tendency to bleeding (nose bleed). Death frequently occurs between three to nine months of age unless liver transplantation is formed. Chronic Tyrosinemia - Children with enlarged liver and spleen, the abdomen is distended with fluid, weight gain may be poor, vomiting and diarrhea occur frequently. Usually develop cirrhosis. Require liver transplant. Homocystinuria Major phenotypic expression. - Ectopia lentils, vascular occlusive disease, malar flush, osteoporosis, accumulation of homocysteine and methionine. Defective activity of cystathionine synthase. Maple syrup urine disease Major phenotypic expression - Overwhelming illness in the first days of life with lethargy progressive to coma, opisthotonus, and convulsions, developmental delay, maple syrup odor, branched chain aminoacidemia and aminoaciduria. Albinism - Deficiency in tyrosinase will result in loss of hair and skin pigments. Individuals with phenylketonuria can have light skin and hair at birth because of low levels of tyrosine. Diagnostic techniques 1) Ferric chloride test 2) Ninhydrin paper chromatography 3) Guthrie bacterial inhibition assay 4) Quantitative measurements of amino acids in plasma and urine 5) GC for urine organic acid analysis Diseases of Carbohydrate metabolism disorders 1) Diabetes Mellitus - Type II due to pancreatic disease (Cushing’s syndrome, abnormality of insulin receptors) Insulin dependent DM (15%): due to destruction of insulin-producing beta cells by autoimmune - no insulin. Caused by environmental factors such as viral infection. Non-insulin dependent DM (85%): due to peripheral tissues resistance to the action of insulin. Clinically related to obesity, glucose is unable to enter tissue cells without the presence of insulin. Urine Examination 1. Glycosuria - Detect the presence of glucose in urine - Glucose does not exist in urine until glucose plasma levels exceed > 10 mmol/L 2. Ketone in urine/plasma - Ketone bodies such as acetone, acetoacetate and B-hydroxybutyrate accumulate in plasma - Diagnostic to ketoacidosis (without insulin, increased lipid and proteins breakdown with increased hepatic gluconeogenesis and the disorder of glucose entrance into cells) Blood glucose level Examination Need to use tubes that contain fluoride-glycolysis inhibitors, and test stips that contain glucose oxidase enzymes. FBG is better than RBG for diagnostics. FBG < 6 mmol/L (NON-DIABETIC) FBG 6-8 mmol/L (BORDERLINE) FBG > 8 mmol/L (DIABETIC MELLITUS) Oral glucose test: glucose level increased > 11 mmol/L after 2 hours (DM) Treatment 1. Increased liquid intake - ketoacidosis case 2. Injection of insulin 3. Hypoglycemic tablet 2) G6PD Deficiency - Abnormalities in the Pentose phosphate pathway - Oxidation of G6P to GPG does not occur, and no reduction of NADP to NADPH. - No production of NADPH by RBC - no reduction of glutathione, free radicals cannot be controlled, RBC membrane - hemolysis occurs. G6PD Diagnosis 1. Light microscopy: formation of Heinz bodies and fragmentation of RBC 2. Quantitative assay: measuring the reduction of NADP+ to NADPH using a UV spectrophotometer. 3. Screening: fluorescent procedure 3) Von Gierke Disease - Glycogen storage abnormalities - Few glucose is produced, and lactate is used inefficiently against glucagon stimulation - Increased degradation of purine in the liver - Molecular defects in G6P and G-6-translocase deficiency in liver, kidneys, and intestinal mucosa. - Symptoms: hypoglycemia, hyperlipidemia, hyperuricemia (gouty arthritis) - Treatment: need to be fed CHO diets throughout the day 4) Fructosuria - hepatic fructokinase deficiency - Hepatic fructokinase, also known as ketohexokinase (KHK) is the first enzyme that catalyzes fructose from diet to fructose-1-phosphate in fructose metabolism. - Glycine to Arginine, Alanine to Threonine. Disorders of Lipid Metabolism Lipid profile test (10 hours overnight fasting blood sample is required) Screening samples: - Finger prick, fast results - Use test strip, not comprehensive Diagnostic samples: - Venous blood, use reagents - Comprehensive test, reliable Factors affecting the reliability of test: 1. High fat meal the night before the test 2. Physical stress (exercise, infection, surgery) 3. Medication (lipid lowering drugs, corticosteroids) 4. Alcohol intake 5. Pregnancy Optimal blood lipid values Triglycerides : < 1.7 mmol/L Total cholesterol : < 5.2 mmol/L LDL-C : < 2.6 mmol/L HDL-C : > 1.6 mmol/L Dyslipidemia Defined as either one or a combination of the following lipid levels: - TG > 1.7 mmol/L - TC > 5.2 mmol/L - LDL-C > 3.4 mmol/L - HDL-C < 1.0 mmol/L in males or < 1.2 mmol/L in females Primary Dyslipidemia - Single or multiple gene mutations that affect a) LDL receptor b) Lipoprotein lipase enzyme c) ABCA-1 transporter d) CETP molecule - Result in overproduction or defective clearance of lipids - Manifested as very high levels of cholesterol and TG in the blood - Fredrickson classification (patterns of lipoproteins) Familial Hypercholesterolemia - type IIa, autosomal dominant - Heterozygous: Increased LDL 2x (onset 30-40 yrs) - Homozygous: Increased LDL 5-7x (onset ~20 yrs) - Expression of LDL receptors decreased lead to excess LDL in circulation, hence hypercholesterolemia - signs: tuberous xanthomas in tendons, under skin Familial Hyperchylomicronemia - type I, autosomal recessive (RARE) - also known as familial lipoprotein lipase deficiency - mutations in LPL gene - massive accumulation of chylomicrons in the circulation, hence chylomicronemia - signs: creamy top layer in plasma, eruptive xanthomas Secondary Dyslipidemia Caused by underlying diseases such as T2DM, obesity, insulin resistance, chronic inflammation. Atherogenic Dyslipidemia is a typical pattern in T2DM and obese person: HIGH TG, LOW HDL, HIGH small LDL Biochemistry of Gastrointestinal disorder Gastritis Inflammation to the gastric mucosa, which can lead to gastric ulcer. Common causes: Helicobacter pylori infection, irritants (alcohol,smoking), and Zollinger-Ellison syndrome H. pylori - causes acute and chronic gastritis - disrupts the gastric mucosal barrier - it can survive in stomach acid bcs it secretes urease enzyme that neutralizes acid Screening test for H. pylori Urea breath test: capsule containing 14C urea. If H. pylori is present, urease enzyme breaks down the urea, releasing 14C. The blood carries the carbon to the lungs where the patient exhales it. Stool Antigen test: detect H. pylori antigens present in a stool. Pancreatitis If inflamed, exocrine (digestive enzymes) functions are affected Acute Pancreatitis - acinar cells contain various zymogens - premature activation of zymogens leads to inflammation and auto-digestion. - release of digestive enzymes in the blood (amylase, protease, lipase) - main causes: alcohol abuse, gallstones WBC, TG, glucose, bilirubin, Alanine aminotransferase, C-reactive protein INCREASES. Chronic Pancreatitis - acinar cell damage due to chronic inflammation and fibrosis - formation of scar tissue causes dysfunction of acinar cells, lead to low digestive enzymes - common symptom: Steatorrhea (fatty stool) Indirect test: fecal elastase-1. Elastase-1 levels in chronic pancreatitis are LOW. Intestinal Disorders Malabsorption Syndrome (FAT) Caused by bile duct obstruction, chronic pancreatitis, celiac disease. - Characteristic: Steatorrhea. Test: Sudan stain test Sudan stain test: Positive (lipid presents, with two layers). Negative (lipid absent, with one even layer) Malabsorption syndromes 1) Triolein breath test: fat malabsorption 2) Hydrogen breath test: CHO malabsorption 3) Xylose absorption test: CHO malabsorption 4) Schilling test: vitamin B12 malabsorption GI Bleeding Upper GI bleed: originates from the esophagus to the ligament of Treitz (peptic ulcer, duodenal ulcer, malignancy) Upper GI bleed screening: Black, tarry stool (melena). Due to digestion of hemoglobin in intestines. (50 ml or more blood loss to turn the stool black) Lower GI bleed: originates from the colon, rectum, or anus (diverticulosis, hemorrhoid, anal fissure, malignancy) Lower GI bleed screening: Hematochezia (fresh bloody stool). - Fecal occult blood test (FOBT) - Stool DNA test (detect abnormal DNA associated with colorectal cancer or precancerous polyps) (KRAS, TP53 genes)