D. Significance of Testing Drugs of Abuse ai zi 3. Cocaine ‘+ Alkaloid found in a plant, Erythroxylon coca ‘+ Medically used as a local anesthesia an vasoconstrictor in nasal surgery and to dilate pupils in ophthalmology + Available in two forms: 1. powder (hydrochloride salt) = administered through nasal insufflation or snorting 2. crack (free-base product) = rock crystal that is heated and smoked; term refers to crackling sounds heard when itis heated = Benzoylecgonine is the primary metabolite of coc © CNS stimulant Lysergic Acid Drugs (LSD) «Structurally similar to serotonin ‘| Synthesized from a naturally occurring ergot alkaloid found in the fungus, Claviceps purpurea which grows in wheat and other grains * Psychedelic drug, hallucinogen "Dosage forms include: tablet, gelatin, powder, capsule ‘Amphetamines * Associated with sympathomimetic syndromes "Stimulants and hallucinogen * Include amphetamines and methamphetamines * Designer amphetamines (also known as designer drugs or club drugs) are derivatives of amphetamine. This includes: phenylethylamines, benzylpiperazines, phenylpiperazines and pyrrolidinophenone = Representative example is ecstasy (MDMA - methylenedioxymethamphetamine) Peeters ca ec Drug-facilitated sexual assault = Includes: anti-cholinergics, antihistamines, barbiturates, benzodiazepines, chloral hydrate, dextromethorphan, ethanol, gamma-hydroxybutyrate, ketamine, opioids, sedative-hypnotics Mlicit drug use during pregnancy - Includes: cocaine, opiates, cannabinoids, amphetamines, etharfol, PCP “Doping” in athletic competitions - Drugs that may cause performance enhancements, but may endanger the athlete's health - Testing for athletes include: > stimulants, narcotics, cannabinoids, gucocorticosteroids > Anabolic-androgenic steroids, hormones, hormone modulators, oxygen transfer enhancement CLINICAL CHEMISTRY: "Working hard for something we don’t care about is called stress. Working hard for something we love is called passion” - AnonymousL CARBOHYDRATES What are Carbohydrates? A. Characteristics of Carbohydrates * composed of three elements carbon, hydrogen and oxygen «hydrates of carbon; general formula: Cx(H20)y «water soluble; most are reducing sugar, except for SUCROSE Functions of Carbohydrates = Major component of human diet, an important source of body energy * Found as part of the cell membrane * Found as antigens in RBCs to determine blood type (ABO blood antigens) C. Classification of Carbohydrates = Ribose and deoxyribose are part of RNA and DNA respectively. * Fructose is also known as levulose or the fruit sugar # Lactose is known as the milk sugar. It is found in dairy products (milk and cheese}! /)sSucrose is known as the common table sugar. It is obtained from beets and sugar cane. 4 Maltose is found in cereals, wheat and malt products. +) Glycogen is the storage form of glucose in the body. It is stored in the liver and skeletal muscles Prone (aeret tints MONOSACCHARIDE DISACCHARIDE Pentose (5 carbon) Sucrose Glucosans - ribose, deoxyribose = fructose + glucose (starch, glycogen) Hexose (6 carbon) Maltose Fructosans (inulin) - glucose, galactose, = glucose + glucose Cellulose fructose Lactose Chitin = galactose + glucose D. Glucose (Dextrose) * Glucose is the principal and almost exclusive carbohydrate circulating in the blood * Glucose is the central, pivotal point of carbohydrate metabolism «Brain is the most important glucose consumer. CNS consumes about 50% of glucose + used by the body + Glucose can be derived from (1) diet, (2) from body stores like glycogen and (3) from endogenous synthesis from proteins or glycerol of triglycerides| E. Carbohydrate Metabolism 1. Carbohydrate Digestion * Polysaccharides and disaccharides from diet enter the body through mouth * Carbohydrate digestion starts in the mouth through the enzyme salivary amylase (aka ptyalin) * Only partial digestion of carbohydrates occurs in the mouth. * Partially digested carbohydrates go from the mouth to the esophagus and into the stomach * No carbohydrate digestion occurs in the stomach due to the acidic pH. + From the stomach, carbohydrates go toward the small intestines. * Alkaline pancreatic secretions increase the pH of the intestines, enabling carbohydrate digestion through pancreatic amylase (aka amylopsin). Enzymes such as maltase, sucrase and lactase hydrolyzes the disaccharides to form monosaccharides (glucose, galactose and fructose) * Monosaccharides are absorbed from the duodenum and ileum into blood 2. Carbohydrate Metabolism in the Blood * Metabolism of hexoses in the blood can result to: a. Energy production by conversion to carbon dioxide and water b. Storage as glycogen in the liver c. Storage as triglycerides in the adipose tissues d. Conversion to ketoacids, amino acids or proteins F." Regulation of Glucose Concentration in the Blood 1. Processes involved in carbohydrate metabolism * Glycolysis - metabolism of glucose molecule to pyruvate or lactate to energy - decreases blood glucose since glucose is consumed to produce lactate/pyruvate = Gluconeogenesis = formation of glucose-6-phosphate from non-carbohydrate sources = increases blood glucose; new glucoses are formed from other sources + Glycogenolysis = breakdown of glycogen to glucose for use as energy - increases glucose since glycogen is degraded into glucose molecules + Glycogenesis = conversion of glucose to glycogen for storage = decreases glucose since excess glucoses in the body is store in the liver and skeletal muscle as glycogen “Once you have become fearless, life becomes limitless” - Anonymous CLINICAL CHEMISTRY:~ conversion of carbohydrates to fatty acids ~ decreases glucose since carbohydrates are converted into fatty acids and stored as fats * Lipolysis - Breakdown of fats; fats are used as energy 2. Hormones involved in carbohydrate metabolism a, Hormones that increase blood glucose - Glucagon, epinephrine, cortisol, growth hormone, thyro: b. Hormones that decrease blood glucose - Insulin c. Regulator hormone: Somatostatin — inhibits release of growth hormone, insulin and glucagon un Produced by beta cells of pancreas as, proinsulin Proinsulinis converted into insulin through the removal of C-peptide fragment Stimulus: increasein plasma glucose causes an increase in insulin Action: Increases glucose entry into cells . Promotes glycogenesis . Promoteslipogenesis Promotes glycolysis . Promotes synthesis of amino acids ‘from pyruvate “Once you have become fearless, life becomes limitless” - Anonymous ee) Produced by the alpha cells of pancreas Stimulus: decrease in plasma glucose triggers an increase in glucagon Action: a. Promotes glycogenolysis b. Promotes gluconeogenesis CLINICAL CHEMISTRY:I. oan i Specimen for Glucose Determination Whole blood, serum, plasma, CSF, serous fluids and urine can be used as sample Standard clinical specimen is fasting venous plasma. Fasting Blood Sugar should be obtained after 8 to 10 hours of overnight fasting. Venous blood has lower glucose levels compared to arterial blood Capillary blood has higher glucose levels compared to venous blood Whole blood gives approximately 10 to 15% LOWER glucose levels than serum or plasma. To convert whole blood glucose into serum or plasma level, multiply value by 1.15 According to old studies: plasma glucose is 5% lower compared to serum. Recent studies reported that plasma glucose is essentially the same as serum, glucose. Aserum specimen is appropriate for glucose analysis if serum is separated from the cells within 30 to 60 minutes Glucose is metabolized at room temperature at a rate of 7 mg/dL/hr (0.4 mmol/L/hr) At 4°C, glucose decreases by approximately 2 mg/dL/hr. Gray top tubes are preferred for plasma glucose collection. Gray top tubes usually ‘contain sodium fluoride which serves as anti-glycolytic agent, and potassium oxalate which serve as anticoagulant Sodium fluoride binds calcium but is a weak anticoagulant, and thus often combined with oxalate. Sodium fluoride acts as preservative and oxalate acts as anticoagulant (i.e. K+ oxalate). 2mg of oxalate and 2mg of sodium fluoride is often used. It prevents glycolysis for 48-72 hours If NaF is used alone as an anticoagulant, 3 to S times greater concentrations than usual is required (approx. 6 to 10mg of NaF/mL. of blood). 2g of sodium fluoride per mL of whole blood prevents glycolysis for 48-72 hours Fluoride binds magnesium, which causes inhibition of the enzyme enolase. CSF glucose concentration is approximately 60 to 70% that of plasma concentration. Blood glucose should be obtained 1-2 hours before the spinal tap CSF for glucose analysis should be performed immediately. If delay in measurement is unavoidable, the sample must be centrifuged and stored at 4°C or at -20°C. 10% Ree risa RL Relate eat Pata [tet \ by 500mq/dL orIll, Methodologies for Glucose Analysis A. Non-enzymatic or Chemical Methods - 1. Copper Reduction "Most carbohydrates are reducing sugars, meaning, they are capable of decreasing the oxidation state of copper, from the cupric form (Cu>*) into cuprous (Cu) Reducing . Cuz Substances @Cut* (cupric) (glucose) (cuprous) Amount of cuprous ions that forms is directly proportional to the amount of the reducing sugar present. The more cupric ions are reduced to cuprous, the more reducing sugars are present in the specimen = Cuprous can then be quantified using any of the following methods: : a Reagent: phosphomolybdate Positive Result: phosphomolybdenum blue b. Nelson-Somogyi Reagent: arsenomolybdate ] Positive Result: arsenomolybdenum blue € Neocuproine method Reagent: Neocuproine ~ Positive Result: orange-red (oryellow to orange) color =. Copper reduction IS NOT SPECIFIC FOR GLUCOSE since other carbohydrates such as fructose and galactose are also reducing sugars * Sucrose is NOT DETECTED by copper reduction methods since it is a non- reducing sugar 2, Ferricyanide method (Ferric Reduction) oe = Also known as Hagedorn Jensen method; negative/inverse colorimetry = Used in AutoAnalyzers (Technicon) = Reagent: hot alkaline solution of potassium ferricyanide = Reducing sugars can reduce ferricyanide into ferrocyanide ~ = Reduction is accompanied by disappearance of color: from yellow into colorless, disappearance of color is measured at 400nm = Reduction in the color is related to the glucose concentration a = Non-specific for glucose since other reducing sugars can also react 3. Condensation Method (o-toluidine method/ Dubowski method) = Reagents: o-toluidine + glacial acetic acid + 100°C heat * Positive Result: bluish green (or green) color measured at 620-630nm CLINICAL CHEMISTRY:esa) B, Enzymatic Methods 1. Glucose Oxidase Step 1: Glucose oxidase Step 2: a glucose oxidase GLUCOSE + OXYGEN GLUCONIC ACID + H202 Polarographic Method - Measures oxygen depletion through electrodes - For each molecule of glucose that reacts, one molecule of oxygen is consumed. - The rate of decrease in oxygen is proportional to the concentration of glucose - Source of error: H20z, ifleft alone, can be broken down to form oxygen, leading to erroneous results. To prevent this, hydrogen peroxide should be destroyed by: 4. Adding ethanol to hydrogen peroxide to form acetaldehyde 4 Adding iodide to form molecular iodine Colorimetric Method - Reagents: Glucose oxidase + Peroxidase + Chromogen/Indicator peroxidase ae H202 + chromogen + H20 + oxidized chromogen - Chromogens/Indicators o-dianisidine (positive blue) aminoantipyine (positive rose-pink) o-tolidine o-anisidine 3-methyl-2-benzothiazolinone hydrazone & N,N-dimethylaniline 4-aminophenazone - Glucose oxidase is specific to the beta-D-glucose A Two forms of glucose exists in the body: alpha-D-glucose = 35-36% beta-D-glucosé = 64-65% 4. Glucose oxidase can only detect for the presence of beta-D-glucose A. Addition of mutarotase can convert alpha-D-glucose to beta-D-glucose - Sources of Error: A. Oxidizing agents (bleach/detergent) causes false increase (false positive) 4. Reducing agents (ascorbic acid, uric acid, bilirubin, hemoglobin) causes false decrease (false negative) - Addition of potassium ferrocyanide decreases interference with bilirubin a A a 4. diethyl-p-phenylenediamine a a A CLINICAL CHEMISTR' “Once you have become fearless, life becomes limitless” - Anonymousa Hexokinase with Glucose-6-phosphate dehydrogenase Reference method for glucose analysis Step 1: GLUCOSE + arp tomes, GLUCOSE-S-PHOSPHATE + ADP Step 2: GLUCOSE-6-PHOSPHATE gp _6-PHOSPHOGLUCONATE + —_— + NAD or NADP NADH or NADPH Absorbance of NADH or NADPH is measured at 340nm Increase in absorbance is proportional to the amount of glucose present NAD is required if the G6PD used is obtained from bacteria (Leuconostoc mesenteroides) NADP is required if the G6PD used is obtained from yeast Product of the G6PD reaction is 6-phosphogluconate (or 6- phosphogluconolactone in some references) ‘The specificity of this enzymatic system does not lie on the use of hexokinase Since hexokinase reacts with any hexoses (glucose, galactose, fructose). Tris the G6PD that makes the reaction specific for glucose determination Some modification adds an additional colorimetric step. NADH or NADPH from the 2" step can be reacted with a chromogen such as phenazine methosulfate with nitrophenyl tetrazolium derivative. Absorbance is then measured at 520nm. Sources of Error: hemolyzed and icteric sample can cause a false decrease Glucose dehydrogenase Variations a. Spectrophotometric Step 1: 2 alpha-D-glucose MUtarolase, neta D-glucose Step 2: lucose dehydrogenase beta-D-glucose + NAD Zeese ehyeo9er8®*, 1 cluconolactone + NADH Step 3: (MTT is a color reagent) MTT + NADH 22282282, wTTH (blue) + NAD E i é Z g drecta b. Through Electric current: Step 1: Glucose + Pyrroloquinoline quinone (PQQ) glucose dehydrogena z glucose dehydrogenase, Giyconolactone + PQQH; Step 2: PQQH) + ferricyanide —» PQQ + ferrocyanide +2H= Step 3: ferrocyanide — ferricyanide + 2e~ = Glucose dehydrogenase is derived from bacteria (Bacillus cereus) = Highly specific for glucose, shows no interference from common anticoagulants and from substances normally found in serum - Sources of error: maltose, icodextrin and galactose can cause false increase IV. _Methodologies for Other Carbohydrates A. Seliwanoff/ Selivanoff's Test for FRUCTOSE = “Reagent: hot hydrochloric acid and resorcinol ss Positive result: red within 30 seconds B. Bial’s Test for PENTOSES ~ Reagent: Hydrochloric acid, orcinol and ferric chloride =. Positive result: green V. Laboratory Tests A. Random blood glucose - blood glucose taken any time ofthe day and without any fasting; often used for emergency cases B. Fasting blood glucose ° - taken after at least 8-10 hours of overnight fasting; usually done in the morning, to prevent the effects of diurnal variation caused by hormones CATEGORIES OF FASTING PLASMA GLUCOSE Normal fasting glucose = <100mg/dl. Impaired fasting glucose/Prediabetes = 100-125 mg/dl. Provisional diabetes diagnosis = >126mg/dl. Ses pao ae ia Weare iar oraE iG Scene Oral Glucose Tolerance Test ORe\cum arcuate) to consume a normal to Pele eRe eee Reece cms Ee A ac! - aseries of glucose testing; a fasting blood glucose is determined after an 8-10 hours of fast = glucose load is given, and a series of blood samples for glucose assays are then collected 30 minutes, 1 hour and 2 hours after glucose load intake carbohydrates per day) for 3 days prior to the test Patient should discontinue, if possible, medications known to affect glucose tolerance Patient is asked to fast overnight and to avoid excessive physical activity. Patient should fast at least 8-10 hours but not greater than 16 hours. OGT testing should be performed on the morning to prevent hormonal diurnal effect on glucose. Blood glucose is taken every 30 minutes for 2 hours. Patient should be ambulatory. Patient should refrain from exercise, eating or drinking (except water) and smoking 6. Fasting blood glucose is measured before giving the glucose load; if the fasting glucose of greater than 140, test should be terminated; ifthe fasting glucose is less than 140mg/dL, glucose load should be given to the patient 7. Glucose load for an adult is 25g. Children receive 1.75g per kg of body weight, max of 75g, 8, Patient should finished drinking the glucose load within S minutes |. Patient should not vomit. If patient vomits, discontinue the test.CHECKPOINT! Clinic D. 2-hours Post Prandial Blood Glucose ~ fasting blood sugar is initially, patient is then given glucose load (usually 75g) andi plasma glucose is determined after 2 hours = normally, blood glucose levels should be back near the reference limits approximately 2 hours post load CATEGORIES OF 2-HOURS PLASMA GLUCOSE Normal glucose tolerance = <140mg/dL © Impaired glucose tolerance/Prediabetes = 140-199mg/dl, Provisional diabetes diagnosis = =200mg/dL E. Glycated (glycosylated) hemoglobin - used for long term monitoring of glucose control over the previous 2-3 months (18-20 weeks period in some references), dependent on the lifespan of RBCs (120 days) ~ itis formed by attachment of glucose at the end or both N-terminal valines of, the beta chains of normal adult hemoglobin © for every 1% increase in HbA1c, there is a 35mg/dL (2mmol/L) change in plasma glucose. - patients with shorter RBC lifespan such as in cases of hemolytic anemia, will have decreased glycated hemogiol -_ specimen is EDTA-whole blood = values can be determined by (1) assays based on structural differences, such as affinity chromatography (2) based on differences on electrical charges, such as in ion-exchange chromatography; electrophoresis and high-pressure liquid chromatography - HPLC can measure all glycosylated hemoglobins (HbA1a, HbA1b, HbA1c) - labile Pre-HbA1c (also known as aldimine) interferes with isoelectric focusing - Affinity chromatography is NOT affected by other hemoglobins and temperature e - ideal value for HbA1c is less than 7% De) Wetter LONG TERM GLUCOSE [rel evel eae aa Penne eet ard level CLINICAL CHEMISTRY: ‘Once you have become fearless Ife becomes limitless inonymaus,CHECKPOINT! C . Fructosamine (Glycated Albumin) - used for monitoring glucose control over the previous 2 to 3 weeks - albumin has a life span of 20 days in the circulation; - affected by changes in albumin levels; patients with hypoalbuminemia has decreased glycated albumin results . AGE (Advanced Glycation End-products) - Non enzymatic attachment of ghicose to long-lived proteins, lipids or nucleic acid can produce Amadori early-glycated products - These undergo a series of rearrangements, dehydration and fragmentation reactions resulting in the stable AGEs - AGEs are irreversible and they accumulate continuously - Patients with diabetes mellitus have more AGE than healthy subjects Clinical Significance of Glucose Results Hypoglycemia + _ Decreased glucose levels = Glycemic factors such as glucagon are released when glucose levels reach 65: Zomg/di. -1| Observable signs and symptoms of hypoglycemia appear when glucose levels re ‘50-55mg/dL. - Symptoms of hypoglycemia can be classified as: (1) Neurogenic - triggered by autonomic nervous system; include trembling, sweating, nausea, rapid pulse, lightheadedness, hunger and epigastric discomfort (2) Neuroglycopenic - caused by diminished glucose supply to the central nervous system; include headache, confusion, blurred vision and dizziness to seizures, loss of consciousness and even death : - Critical value for glucose is 40mg/dL; excessively low glucose values can cause severe CNS dysfunction especially if blood glucose value drops to 20-30mg/dL. - Whipple's Triad is used to assess patients with episodes of low plasma glucose levels. This refers to symptoms consistent with hypoglycemia associated with documented low plasma glucase level and relief of symptoms with correction of hypoglycemia - Random blood sugar can be used in cases of insulin shock CLINICAL CHEMISTRY: “Once you have become fearless, life becomes limitless” - Anonymousencoun ukeln Bae Hypoglycemia can be: (1) Reactive (postprandial) ~ caused by a stimulus; not usually related to underlying disease (2) Fasting (post-absorptive) - often related to an underlying disease = Causes of Hypoglycemia include: (1) Drug-induced (e.g. insulin, alcohol, etc) (2) Hormonal deficiencies (e.g. glucagon, epinephrine, cortisol, growth hormone) (3) Endogenous Hyperinsulinism (e.g. insulinoma) (4) Autoimmune hypoglycemia (e.g. insulin antibodies or insulin-receptor antibodies) (5) Hypoglycemia of Infancy and Childhood (e.g. glycogen storage diseases) (6) Alimentary Hypoglycemia (e.g. post gastric bypass surgery) (7)Idiopathic or Functional Postprandial Hypoglycemia B. Hyperglycemia - Laboratory Findings in Hyperglycemia * Increased plasma glucose levels + Positive urine glucose ~ Urine glucose becomes positive when plasma glucose levels exceed the renal threshold limit or value for glucose ~ Renal threshold limit for glucose is 140-160mg/dL (or 160- 180mg/dL in some references) + Increased urine specific gravity - due to the presence of glucose in urine + Increased serum and urine osmolality (osmolarity) - due to the presence of increased glucose in serum and urine + Ketones in serum and urine (ketonemia and ketonuria) ‘= Decreased blood and urine pH (acidosis) = Electrolyte imbalance Hyperglycemia leads to hyponatremia - Diabetes mellitus is a group of diseases in which blood glucose levels is elevated. This is often associated with polyphagia (excessive hunger), polydipsia (excessive thirst) and polyuria (excessive secretion of urine) DIABETES INSIPIDUS ene slivolves aniesitixenaboraioncss| Increased water excretion Increased water excretion, no | with hyperglycemia hyperglycemia | Polyuria with high urine | Polyuria with low urine specific specificgravity gravity | E i & 0 g 5 o Spal cae ex beer ni rerent) - Criteria for Diagnosis of Diabetes Mellitus* (1) Symptoms of diabetes plus random (casual) plasma glucose concentration 2200 mg/dL (11.1 mmol/L). Random is defined as any time of day without regard to time since last meal. The classic symptoms of diabetes include polyuria, polydipsia, polyphagia, and unexplained weight loss. (2) Fasting plasma glucose (FPG) 2126 mg/dl. (7.0 mmol/L). Fasting is defined as no caloric intake for at least 8 hours. é (3) 2-Hour postload glucose 2200 mg/dl. (11.1 mmol/L) during an oral glucose. tolerance test, using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water. *These criteria should be confirmed by repeat testing on a different day. - Diabetes mellitus vs. Renal Glycosuria 1. Diabetes mellitus = increased blood glucose _ positive urine glucose 2, Renal glycosuri normal blood glucose _ positive urine glucose - Classification of Diabetes Mellitus 1. Type I DM. (eg. immune-mediated or idiopathic insulin deficiency) 2. Type Il D.M. (insulin resistance) frequency [ ‘Age of Onset ‘Any (mostcommonin | More common in children & young adults) | advancingage | ‘Risk factor Genetic, autoimmune, | Genetic, obesity, sedentary heals environmental lifestyle, race/ethnicity, NI hypertension, dyslipidemia, ae a polycystic ovarian syndrome Pathogenesis Destruction of pancreatic No autoimmunity, insulin beta cells, usually resistance and progressive autoimmune insulin deficienc | C-peptide Very low or undetectable Detectable | levels Pre-diabetes ‘Autoantibodies (GAD65, ‘Autoantibodies absent | eee _1A-2, IAA) may be present | | Medication | Insulin absolutely necessary, Oral agénts, insulin therapy multiple daily injections or commonly needed insulin pump Therapyto | None known | Lifestyle (weight loss and prevent or | | increased physical activity); delay onset of | Oral medications can be diabetes helpful Previous Insulin-dependentDM _| Non-insulin dependent DM names_ : Juvenile-onset DM Adult-onset DM Ketoacidosis | Prone to ketoacidosis and | Not prone to ketoacidosis diabetic complications | “Once you have become fearless, life becomes limitless” - Anonymous3. Gestational Diabetes Mellitus (GDM) ~ Glucose intolerance that develops in pregnancy, often caused by hormonal! and metabolic changes ~ Can lead to increased perinatal complications such as respiratory distress, hypocalcemia, hyperbilirubinemia and fetal macrosomia ~ Alarge percentage of women with GDM develop diabetes within § to 10 years ~ Todetect GDM: % One-step approach for high-risk individuals Step 1-OGTT > OGTT two-step approach for average-risk individuals Step 1 ~ Initial Screening (1-hr plasma glucose after 50g glucose load) Step 2- OGTT 4. Other Specific Types ~ Endocrinopathies, genetic defects, pancreatic diseases, drug- or chemical- induced, infections C. Glycogen Storage Diseases (GSDs) “ results from deficiency of specific enzyme that causes an alteration in the glycogei metabolism - “patients exhibit hepatomegaly (due to increased liver glycogen stores) and hypoglycemia (due to inability to convert glycogen back to glucose) = Von Gierke disease is the most common glycogen storage disorder eu pA ge Ia - Von Gierke II- Pompe acid alpha glucosidase/ acid maltase y Ill - Cori-Forbes amylo-1, 6 glucosidase/ debrancher enzyme _! Ra IV- Andersen amylopectinase/ glycogen branching enzyme V=MecArdle muscle phosphorylase ey Vi- Hers liver phosphorylase or phosphorylase kinase NW \ e Vit- Tarui muscle phosphofructokinase XI-Fanconi-Bickel | glycogen transporter 2 0 glycogen synthetase a CLINICAL . “Once you have become fearless, life becomes limitless” - Anonymous ziVil. “Walues to Remember teeta eee een nes D. Inborn Errors or Carbohydrate Metabolism 1. Disorders of Galactose metabolism ~ galactosemia = Galactose-1-phosphate uridyl transferase deficiency * Uridine diphosphate glalactose-4-epimerase deficiency + Galactokinase deficiency 2. Disorders of Fructose metabolism = Fructokinase deficiency (Essential fructosuria) + = Fructose-1-phosphate aldolase (Hereditary Fructose Intolerance) + Hereditary fructose-1, 6-diphosphatase deficiency 3. Disorders of Pentose metabolism + Alimentary pentosuria + L-xylulose reductase deficiency (Essential Pentosuria) 4. Other Urinary Sugars = Lactosuria - found in women during lactation and occasionally towards the end of pregnancy, and in patients with lactase deficiency = Fasting Plasma Glucose: 70 to 100mg/dL = Glycosylated hemoglobin: 4-6% (Henry 21st ed); 4.5-8% (Bishop) = Conversion Factor: 0.0555 * Critical Values: <40mg/dL and >500mg/dL E : i d “Once you have become fearless, life becomes limitless” - Anonymous1. Whatare Lipids? A. Characteristics of Lipids * Body's petroleum industry; contains carbon, hydrogen and oxygen = Water insoluble, non-polar; transported in the body by lipoproteins B. Lipid Metabolism from Diet = During digestion in the intestines, pancreatic lipase cleaves off fatty acids to convert lipidSifit6’ more polar compounds or amphipathic lipids = Triglycerides are converted into monoglycerides & diglycerides; cholesterol esters are transformed into free cholesterol & phospholipids are converted into lysophospholipids first ‘= Amphipathic lipids aggregate with bile acids and form micelles "Micelles are absorbed from intestines into the blood = These micelles are re-esterified into triglycerides and cholesteryl esters which are then» transported by lipoproteins . Fortis of Lipids 1. Triglycerides (Triacylglycerol) % possesses 3 molecules of fatty acids and a molecule of glycerol, which serves as the ‘Yoackbone ~~ hydrophobic and water insoluble; serves as the main storage form of lipid in man (as found {in adipose tissues) © Most triglycerides from plant sources are rich in unsaturated fatty acids and e: as oils, whereas triglycerides from animal sources contain mostly saturated fatty acids and are usually solid at room temperature ‘Triglycerides do not have any charge they are therefore known as neutral fats lipids = serves as: = energy source + as integral part of the cell membrane ‘insulation or shock absorber 2. Cholesterol : ~ unique in that, unlike other lipids, itis not readily catabolized by most cells and, therefore, does not serve as a source of fuel. + serves as: + part of cell membrane = converted in the liver to primary bile acids, such as cholic acid and chenodeoxycholic acid, which promote fat absorption in the intestine by acting as detergents (fat emulsification) + canbe converted by some tissue (adrenal gland, testis, and ovary) to steroid hormones (such as glucocorticolds, mineralocorticoids, and estrogens) + canbe transformed to vitamin D3 in the skin by irradiation from sunlightGtecuonune Tl Li - exists in the body in two different forms: + Cholesterol (cholesteryl) Esters or Esterified Cholesterol ~ accounts to approximately 60-70% of the total cholesterol in the body ~ composed of cholesterol ring and a fatty acid «Free cholesterol - accounts to approximately 30-40% of the cholesterol in the body + unesterified cholesterol; cholesterol ring only, no fatty acids attached Phospholipids ~ _ structurally similar to triglyceride except that is 2 fatty acids and a phospholipid head ‘group is attached to the glycerol backbone instead of 3 fatty acids - Phospholipids in the body: = phosphatidyicholine (70%-75%) = _ sphingomyelin (18%-20%) * _ phosphatidylserine, phosphatidylethanolamine (3%-6%) = lysophosphatidylcholine (4%-9%) + phospholipid head groups are hydrophilic and include: such as choline, inositol, serine) and ethanolamine -__two fatty acids in phospholipid: one fatty acid is commonly saturated and the other Is unsaturated ® “important constituent of cell membrane (phospholipid bilayer), contains a polar and nonpolar regions can be found in the lungs as surfactant during respiration Fatty acids © “building blocks of lipids, hydrocarbon chains with terminal COOH group ~~ usually found in triglycerides, phospholipids and cholesterol - inplasma, they may exist in the free or unesterified form, mast of which is bound to albumin - Forms of Fatty acids: "Saturated - fatty acids with no double bonds * Unsaturated — fatty acids with double bond{s), usually in the cis form, making them ‘more fluid + Trans fatty acid ~ fatty acids with double bond(s) in the trans form poproteins ‘Transports lipids * Chemical Composition of the Different Lipoprotein Classes: ieee Chylomicrons 4 VLDL 4-8 16-22 45-65 | IDL _ntermediate between VEDL and LDL [BDL ig-22| 6-8 45-50 | 4-8 HDL 45-55| 3-5 15-20 2-7 CLINICAL CHEMISTRY: “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough Jr.+ _Apolipoproteins - protein portion of the lipoprotein ‘Apo! HDL | ~ activates LCAT that esterifies cholesterol in plasma ' ~ synthesized in the liver and intestine ~ HDL | ~ May inhibit lipoprotein and hepatic lipases and ___|__ increases plasmatrighyceride ~HDL,CMand | ~ May be acofactor for LCAT; increases during fat a | free in plasma absorption; HDL biosynthesis q ~VLDLandLDL | ~ Very large structural protein; synthesized in liver with lipids of endogenous origin = ~CM | ApoC-l |~CMandVLDL | i ___| _ ester transfer protein ees ApoCGlt |~CMandVLDL | ~ Activates lipoprotein lipase a ~ Deficiency causes reduced clearance of triglyceride- | | rich tipoproteins see ApoC-lll | ~ VLDL, HDL ~ Inhibits lipolysis of triglyceride-rich lipoproteins; | decreases clearance rate of remnant particles | ~ Deficiency causes reduced clearance of triglyceride- | Hn lipoproteins } ApoD | ~ HDL. = Activates LCAT ‘ApoE — | ~CM,VLDL,IDL, | ~ Recognition factor that targets CM and VLDL remnants to hepatic receptor; also binds tocell surface LDL receptors and proteoglycans i | | i j | | remnants and HDL | ~ £-2, 8-3, and E-4 isoforms; E-4 Is associated with | high LDL-C, higher risk of CHD and Alzheimer's | disease; £-2 associated with type 3 _|_hyperlipoproteinemia |= Regulates CETP function | ~ Related to activation of LPL; triglyceride metabolism ~ Cell-aggregating factor in Sertoli cells; inhibitor of : the C5*7 complement complex; beta-amyloid clearance in glial cells; cholesterol trafficking in brain ~ Involved in apoptosis; inked to neurologic diseases like Pick’s and Alzheimer's; also known as lusterin = ApoL. ~ HDL ~ May be linked to reverse cholesterol transport | ApoM ~HDL,CM,LDL, | ~ May be linked to HDL remodeling | I VLDL. ‘Apo(a) | ~ Lp(a) | ~ Homologous to plasminogen, may be | | prothrombotic; Ll ~ bound to apoB-100 by disulfide linkage CLINICAL CHEN “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough Jr.Gtecrouurenmenne + Normal Lipoproteins = Chylomicron + Largest and lightest among the lipoproteins; = 80-95% triglyceride by weight = Transports exogenous triglycerides (triglycerides derived from the diet/ food) = Causes non-fasting lipemia (turbidity of serum in non-fasting patients) = No charge; remains in the origin during electrophore: Very Low Density Lipoproteins - _Pre-beta lipoprotein = Transports endogenous triglycerides (triglycerides produced by the body) ~ Low Density Lipoprotein - Also known as the bad cholesterol; beta lipoprotein + Transports cholesterol from the liver to the cells of the body = Transports majority (75%) of the cholesterol - Directly proportional to the risk of atherosclerosis and coronary heart disease (CHD); higher LDL means higher risk ‘+ High Density Lipoprotein - Smallest and heaviest among the lipoproteins + Fastest towards the anode; alpha lipoprotein; good cholesterol + Inversely proportional to the risk of atherosclerosis and coronary heart ase (CHD); lower HDL means higher risk - Responsible for reverse cholesterol transport; transports cholesterol from. the cells to the liver + Abnormal Lipoproteins = Lpta) - also known as the sinking prebeta lipoprotein + fourid in the LDL density range in ultracentrifugation, but moves in the pre~ beta region during electrophoresis = associated with increased risk for coronary heart disease, cerebrovascular disease, stroke and atherosclerosis; may be prothrombotic and can interfere with normal thrombolysis due to its similarity to plasmuinegen = Beta-VLDL - also known as the floating beta lipoprotein; a VLDL that is richer in cholesterol than in triglycerides = found in the VLDL density range but migrates electrophoretically with or near LDL - _ typically seen in patients with type III hyperlipoproteinemia joprotein with lowest density “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough Jr. CLINICAL CHEMISTRY:erect > Lpx = abnormal lipoprotein that is rich in lipids, primarily unesterified cholesterol = and phospholipids - found in patients with obstructive biliary disease; regurgitation of biliary contents in the bloodstream results in the buildup of LpX - migrates towards the cathode during electrophoresis ‘+ Identification of Lipoproteins = Chromatographic methods (i. gel chromatograph affinity) Immunochemical methods - use of antibody directed towards specific apolipoproteins (ie. anti-apoA1, anti-apo-C, etc.) Electrophoresis (Fredrickson-Levy reaction) separates lipoproteins based on their electric charge lipoproteins carry an electric charge at pH 8.6 ~ lipoproteins can be stained using fat stains: Fast red 7b, Oll red 0, Sudan Black B + Ultracemtrifugation ~ separates lipoproteins based on their densities; reference method for lipoprotein analysis ‘fsedimen /EDBERG UNIT ELECTROPHORETIC MOBILIT ENSITIES OF LIPOPROTEINS 3, ‘OF LIPOPROTEINS (ULTRACENTRIFUGATION) youn Lipoproteins, te =| Chylomicrons 1. Chylomicrons Lipoproteins * ore fpopreteins wD) (GEER) | Pre-p-ipoproteins ae fg Low-density lipoproteins Dt) Baa | «-Lvoprotcins lipoproteins. .4. High-density ie (HDL) From Henry's 22nd ed CLINICAL CHEMISTRY: “Climb mountains not so the world can see you, but so you can see the world” _ -D. McCullough Jr. utizenaon Ill, Methodologies ‘A. Assays for Triglycerides Chemical Method "Methods = Van Handel and Zilversmit method = Hantzsch = Modified Van Handel Zilversmit method "Generalized Steps in the Chemical Method of Triglyceride Analysis STEP 1: Extraction * PURPOSE: Pretreatment of the sample to remove the lipids from proteins REAGENT: organic solvent (e.g. chloroform, isopropanol, diethyl ether) Additional step: Adsorption PURPOSE: to remove non-triglyceride glycerols such as phospholipids, monogiycerides and diglycerides as well as interfering substances such as ghucose (carbohydrates) and bilirubin REAGENT: alumina adsorbent mixture, zeolite, florisil or silicic acid STEP 2: Hydrolysis or Saponification PURPOSE: To cleave triglyceride molecules into fatty acids and glycerol REAGENT: potassium hydroxide in ethanol (alcoholic potassium hydroxide) or sodium methoxide triglycerides + 3H,O —*°#_» glycerol + fatty acids| S STEP 3: PURPOSE: To convert glycerol to a measurable compound REAGENT: oxidizing agent such as sodium periodate (glycerol + sodium periodate (NalO4) —> formaldehyde + formic acid| STEP 4: Colorimetry PURPOSE: Aids in the measurement of the compound that forms after oxidation; _ measured by its absorbance after addition of color reagent REAGENT: color reagent ‘D chromotropie acid & H.SOs = pink chromophore measured at 500-600nm > diphenylhydrazone = product measured at 500-600nm ‘> acetylacetone and ammonia (Hantzsch) = reactant of choice; formation of diacetyl dihydrolutidine, a yellow product that can be medsured either: ~ spectrophotometrically 412nm ~ fluorometrically (Excitation light: 400nm; Emitted light: 485nm) + CDC Reference Method for Triglycerides New reference method is Gas Chromatography Isotope Dilution Mass Spectrometry Original Reference Method: 4. Extraction with chloroform 4. Oxidation with periodate + 2. Adsorption with silicicacid 5, Color development using chromotropic acid 3. Hydrolysis with KOH 6, Absorbance is measured at 570nm CLINICAL CHEMISTRY: “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough Jr.2, Enzymatic Method a, INITIAL ENZYMATIC REACTION (Lipase and Glycerokinase) Triglycerides P22; Glycerol + Fatty acids Glycerol + are AYSEOKINIES GIy cerophosphate + ADP THE INITIAL ENZYMATIC REACTION CAN BE FOLLOWED BY ANY OF THE FOLLOWING COUPLED ENZYMATIC REACTIONS: . = Lipase-Glycerokinase coupled with glycerophosphate dehydrogenase and diaphorase ‘Glycerophosphatg dehydrogenase Dikydroxyacetone phosphate + NADH + H4| |Glycerophosphate + NAD INADH + Tetrazolium dye Dl@POPS6 formazan + NAD* “Measurement of absorbance of: ¥ Absorbance of NADH can be measured at 340nm, after the slycerophosphate dehydrogenase reaction Absorbance of formazan dye can be measured between 500-600nm +, Lipase-Glycerokinase coupled with glycerophosphate-oxidase and peroxidase Glycerophosphate +0, Clvcetophosphats ihyctroxyacetane + HO; oxidase s+ Phenol + 4-aminoantipyrine —etxidase, Quinoneimine dye +2H,0 “Absorbance of the red quinoneimine dye can be measured spectrophotometrically at 500-50Snm_ “Hemoglobin and ascorbic acid can also interfere with the peroxidase step Bilirubin interference can be minimized by adding potassium ferrocyanide to the reaction mixture with peroxidase. Potassium ferrocyanide reduces bilirubin into an inactive compound. * Lipase-Glycerokinase coupled with pyruvate kinase and lactate dehydrogenase ‘ADP + Phosphoenol pyruvate es ATP + Pyruvate Lactate NAOH +H ——_Laclale_, | actate + NAD* Pyruvate + +H adrogenas’ Uacale + \ NAD* can be measured at 340nm spectrophotometrically or ‘+ NAD* can be measured fluorometrically: Excitation light at 355nm Emitted light at 460nm E | z 3 “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough jr.B, Assays for Total Cholesterol 1. Chemical Method = Methods ens Gan Zlatkis, Zak & Boyle | One-step Rapid method; subject to Colorimetry | chromogen interference; color differences between free cholesterol and cholesteryl ester is a problem in the method Carr & Drekter Two-step Chromogen interference and coloi | Extraction | differences between free cholesterol and Colorimetry. cholesterylester are problems inthe Ze method; protein interference removed ‘Abell Three-step Protein interference removed; color Extraction | differences between free cholesterol and Saponification cholesteryl ester is removed | é S | Colorimetry _| Chromogen interference partially removed ‘Schoenheimer/ Four-step | Protein and chromogen interferences were | Sperry Extraction | removed; color differences between free | | saponification cholesterol and cholesteryl ester is Purification removed Colorimetry ‘toeGeneral Steps in Chemical Method of Total Cholesterol Analysis ©. STEP 4: Extraction PURPOSE: cholesterol is separated from protein REAGENT: Bloor’s reagent (composed of ethanol-ether at a 3:1 ratio)/ chloroform/ hexane Additional Step: Adsorption PURPOSE: extracts all forms of cholesterol, leaving behind most of the sterols REAGENT: zeolite STEP 2: Saponification/ Hydrolysis PURPOSE: cholesteryl esters after extraction are hydrolyzed into free cholesterol and fatty acid REAGENT: alcoholic potassium hydroxide (KOH) STEP 3: Purification: PURPOSE: to precipitate free cholesterol; removes error of nonspecific chromogen interference REAGENT: digitonin ~ measure cholesterol before and after digitonin treatment to determine cholesterol fraction + STEP 4: Colorimetry PURPOSE: color development, measured spectrophotometrically REAGENT: color reagent CLINICAL CHEMISTRY: “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough Jr.ceo) Cad Reagent ‘Acetic anhydtrid End product Cholestedienyl monosulfonic acid | End color’ | Green (not stable; add sodium sulfate | | to stabilize) measured at 410nm | (Calbreath) or 620nm (Tietz) Sulfuric acid + Ferric iron | Cholestedienyl ~-Red(stable) Furie acid isulfonic acid + CDC Reference Method for Cholesterol (Abell, et.al.) Hydrolysis with alcoholic potassium hydroxide Extraction with hexane Extract is dried in vacuo Dried extract is then treated with Liebermann-Burchard regent (Acetic acid + acetic anhydride + sulfuric acid) Read at 620nm after 30 minutes Seto 2, Enzymatic Method a. Spectrophotometric as»Absorbance of quinoneimine dye is measured at 500nm = _ 4-aminoantipyrine is also known as 4-aminophenazone = “Some methods use methanol on the third step instead of 4-aminoantipyrine Methanol, in the presence of peroxidase is oxidized into formaldehyde. Formaldehyde is then detected using acetylacetone (similar to colorimetric step of triglyceride chemical method) = Some methods measure the absorbance of cholest-4-en-3-one at 240nm Cholesteryl ester . H, Fi Cholesteryl ester + HO eae sey, Cholesterol + Free fatty acid Cholesterol +O, Cholesterol Cholest-4-en-3-one + H,0; oxidase 2 H,O; + Phenol + 4-aminoantipyrine Peroxidase, E Quinoneimine dye + 2H,0 b. Oxygen consumption ~ Uses cholesteryl ester hydrolase and cholesterol oxidase - Hydrogen peroxide that forms is mixed with peroxidase, which causes the formation. of water and oxygen = _ oxygen electrode measures the oxygen released after the decompo: peroxide ~ not easily automated and generally require a lot of cholesterol oxidase é $ 4 CLINICAL CHE! “Climb mountains not so the world can see you, but so you can see the world” z =D. McCullough jr. =ftizetaoa ©. Standing Plasma Test \ Chylomicrons, if present in appreciable quantities are detected using the standing plasma test \ 2m of plasma is placed in 10 x 75mm test tube and allowed to stand in the refrigerator at 4°C undisturbed overnight ‘= Chylomicrons appear as “floating cream layer” visually Chylomicrons in fasting plasma is considered abnormal Plasma sample that remains turbid after standing overnight indicates excessive amounts of VLDL. £ D. Methods for HDL cholesterol quantitation: 1. Precipitation + VLDL and LDL is precipitated by adding precipitating agents (polyanion-divalent cation) suchas: = Heparan sulfate-Mn“ (Heparin-Manganese chloride) F + Dextran sulfate-magnesium chloride = Phosphotungstate-magnesium chloride (sodium phosphotungstate-Mg"*) = Heparin-Ca’* CDC reference method (4) ultracentrifugation - to remove VLDL e (2) precipitation with Heparin- Manganese chloride - to remove LDL, (8) measurement of the supernate using Abell-Kendall method 2. linmunoassays SP ELisa + ‘Immunonephelometry E, Methods for LDL cholesterol quantitation: 1. Calculation * triglycerides ) 5 ¥_ "TAG/S = VLDL" is applicable only if TAG is <400mg/dL - ¥ “TAG x 0.16 = VLDL" is applicable if TAG is >400mg/dL = Y Friedewald * (TAG divided by 5) is used if the unit used is mg/dL. = (TAG divided by 2.175) is used if the unit use is mmol/L 7] ¥ DeLong * (TAG divided by 6.5) Is used ifthe unit used is mg/dL = |" (TAG divided by 2.825) is used if the unit use is mmol/L ILDL = total cholesterol - HDL - ( “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough Jr.Assays for Phospholipids Chemical Method + Extraction Oxidation: converts phospholipid phosphorus into inorganic phosphorus Reagent: concentrated acid (wet-ash oxidation) + Colorimetry Phosphorus is mixed with molybdate reagent to form phosphomolybdate ion Phosphomolybdate ion is then subjected to reduction to form inolybdenum blue id mass + Total phosphorus x 25 = Phosphi Enzymatic Method “Phospholipase D: Phospholipid is hydrolyzed using phospholipase D into choline + Choline oxidase: Choline is then oxidized into betaine and hydrogen peroxide Peroxidase: Hydrogen peroxide is then mixed with phenol and 4-aminoantipyrine to form quinoneimine dye, which is measured at 500nm Assays for Fatty acids + Perform lipid extraction ‘Wash with dilute sulfuric acid to remove contaminants Titrate purified extract with dilute NaOH with thymolphthalein as indicator ‘Specimen Considerations and Patient Preparation: Fasting requirement of atleast 12 hours - patient can drink water, but no food should beconsumed. Non fasting state can affect triglyceride but cholesterol is not significantly affected Ethanol consumption must be restricted at least 2 days prior to the test since it can cause an increase in triglyceride Serum or EDTA-plasma may be used (citrate and fluoride can alter osmolarity; heparin interferes with electrophoresis); ona EDTAis preferred for lipoprotein assays © Bay anteeraniocer eer plead citer snermense ke paroled (presence of bilirubin levels greater than Smg/dL can eect interfere) Avoid water contamination, especially for cholesterol assays (Lieberman Burchard) Acceptable CV for Lipid analysis: Total Cholesterol = 3% HDL-cholesterol = 49% LDL-cholesterol = 4% TAG=5% ete “Climb mountains not so the world can see you, but so you can see the world” ~ D, McCullough Jr.Ginnie V. Clinical Significance A. Arteriosclerosis vs Atherosclerosis Arterlosclerosis = isageneral term for the thickening and hardening of arteries, Atherosclerosis - isatype of arteriosclerosis - hardening of the arteries, caused by plaques that build up inside the arteries = Plaque is made of cholesterol, fatty substances, cellular waste products, calcium and fibrin B. Coronary Heart Disease = Refers to the broad spectrum of heart disease resulting from impaired coronary blood flow - Clinical (Non-Laboratory) Risk factors for CHD Cigarette smoking (any smoking in the past month) Hypertension (blood pressure >140/90 mm Hg or on antihypertensive medication) Family history of premature CHD (CHD in male first-degree relative <55 years, or in female first-degree relative <65 years) ‘Age (men >45 years; women >5S years) Obesity Diabetes mellitus “= Sedentary lifestyle ¢. Analphalipoproteinemia ~ also known as Tangier disease; deficiency of HDL D. ABetalipoproteinemia ~ also known as Bassen-Kornzweig syndrome; deficiency of apoB (B48 and B100); notable acanthocytes in peripheral blood smear E, Fredrickson and Levy's Hyperlipoproteinemia Phenotypes Blood Lipoprotein Patterns in Patients with Hyperlipoproteinemia Peat t G0 616 Extremely el Ha__| Elevated LDL. a Hib Elevated LDL and VLDL mt (_w__| Elevated VLDL | Elevated VLDL and presence of chylomicrons to the presence of chylomicrons ir: RUMEN ae RO |_1 | Creamy layer on clear plasma Ha _| Clear = Mb _| Clear or slightly turbid [77 Clear or slightly turbie V_| Clear or turbid_ | V_[ Creamy layer on turbid plasma “Climb mountains not so the world can see you, but so you can see the world” =D. McCullough Jr. BH CLINICAL CHEMISTRY:Girne nein F, Lipid Storage Diseases nan i ae ‘abry's disease alpha galactosidase cae gangliosidosis beta galactosidase Gaucher dase Krabbe je beta galactosidase Niemann Pick sphingomyelinase a Metachromaticleukodystrophy | arylsulfatase A Sandhoff total hexosaminidase (hexbsami Nay Mere hexosaminidase A ake = VI. Values to Remember A. ATP III Classification for LDL, Total and HDL Cholesterol, and Triglyceride values (Henry 21% ed) Eas primal [<150 mg/dl Normal | <100 mg/dl. | 100-129mg/ai. jcar optimal/above optimal | | 150-199mg/dl._ Borderline high 130-159mg/aL Gee | 200-499me/dL High ~~ ——«*Y | 200-499mg/di. High | [B500mg/al Very high desirable | <40 mg/dl. low borderline high | 260 mg/dL shigh B. Reference ranges: Total cholesterol= _140-200mg/dL. (Male) 29 to 60mg/dl. (Female) 38-75mg/dL 57 to 130mg/aL. Triglycerides = 67 to 157mg/dL. €. Conversion factors - Cholesterol (mg/dL. to mmol/L) 0.026 ‘Triglycerides (mg/dL tommol/L) = 0.0113 Borly Mass Index below 18.5 = underweight 18.5 to 24.9 =healthy 25 to 29.9 = overweight 30 or higher = obese Piuciousstenon | F 5 g o “Climb mountains not so the world can see you, but so you can see the world” = D. McCullough Jr.L What are Proteins? A. Characteristics of Proteins Most abundant macromolecule in the body The only macromolecule expressed in grams per deciliter, unlike lipids and carbohydrates, which are both expressed in milligrams per deciliter Contains carbon, hydrogen, oxygen and nitrogen Nitrogen is the element that differentiates protein from other macromolecules - lipids and carbohydrates do not contain nitrogen Mostly synthesized in the liver, except for some proteins (Le. immunoglobulins which are produced by plasma cells) Water soluble; Amphoteric - can be positively charged or negatively charged Alkaline pH (predominance of OH: ions) proteins are negatively-charged Acidic pH (predominance of H* ions) proteins are positively-charged Amino acids are the building blocks are proteins. Amino acids have an amino group (- NH;) and a carboxylic acid group (-COOH) Peptide bonds (also known as amide linkages) hold amino acids together Essential amino acids are amino acids that are important for protein synthesis. They ‘re not produced in the body and are therefore “essential” constituent of the diet Essential amino acids include: Y Isoleucine ¥ Leucine Y Lysine Y Methionine Y Phenylalanine ¥ Threonine ¥ ‘Tryptophan ¥ Valine Structure of proteins: Primary structure - refers to the sequence of amino acids in a peptide or protein © Secondary structure - may include the alpha-helix, beta-sheet, beta-turn or random structure of proteins \ Tertiary structure - folding of proteins into a three-dimensional shape; denaturation of proteins refers to unfolding that occurs when téinperature change or in the presence of organic solvents, detergents or reagents that may change the tertiary structure Quaternary structure — incorporation of two or more polypeptide chains or ‘subunits into a larger unit; example include creatine kinase with two subunits as ell as LDH and hemoglobin having four subunits+ Proteins can be differentiated based on: \ Differential solubility - proteins are affected by pH, lonie strength, temperature and dielectric constant. Isoelectric point is the pH where proteins have no net charge. Zwitterion is an ion that has two differing charges but the net charge on the molecule is zero. “Molecular size - can be accomplished through ultracentrifugation or dialysis \ Molecular mass - can be accomplished through mass spectrometry Electrical charge - can be accomplished using fon-exchange chromatography and electrophoresis. \ Surface adsorption - can be done using chromatography * Plasma vs Serum protein - plasma contains fibrinogen whereas serum does not; an approximate 4% decrease in total protein content in serum due to absence of fibrinogen B, Protein Metabolism + Protein digestion begins in the stomach and completed in the small intestines + High acidity denatures the protein making them susceptible to enzyme digestion + Pepsin Is an enzyme in the stomach responsible for digesting proteins into amino acids +" Amino acids are absorbed from the small intestines into the blood and then ‘Fansported to the liver . Functions of Protein *y/ Regulate colloidal oncotic pressure + laccas carrier for transport of different substances FS Coagulation cascade + “Complement fixation + Serves as biocatalysts or enzymes + Maintenance of acid-base balance (proteins act as buffer) functions (antibodies and complement proteins) + Form many important intracellular and extracellular structures + Important for tissue repair (collagen) . * Generate energy through catalysis and electron transfer * Produce motility through contractile elements a + Assemble molecules = Immunolo; + Serveas ion channels and pumps = Serve as receptors, hormones, and cytokines for intercellular regulation * Constitute signaling networks for intraceltular regulation jever pul off Til tomorrow the book you can read today” — Holbrook Jackson EaTecan) Major Plasma Protein | Prealbumin Prealbumin a [Albumin | Albumin & [‘Alpha, globulin Alpha, antitrypsin, alpha-fetoprotein,alpha-lipoprotein, alpha-{-acd | glycoprotein, alpha-1-anti-chymotrypsin, inter-alpha-trypsin | inhibitor, Ge-globulin es [Alphas globulin | Cerufoplasmin, haptoglobin, alphaz macroglobulin, jetaglobulin | Transferrin, hemopexin, beta-2 bulin, | nicroglobulin, complement system, | fibrinogen, LDL, VLDL, C-reactive protein [Gamma globulin | Immunoglobulins, Creadtive protein (in some books) E, Proteins and their Specific Functions > PREALBUMIN “also known as transthyretin, migrates anodal (faster) than albumin “204 most predominant protein in the CSF functions as a carrier protein for thyroid hormones and vitamin A ALBUMIN protein that is present in highest concentration in the plasma <> synthesized by the liver ‘derived from the Latin name “albus” which means white, originated from the white precipitate formed during the boiling of acidic urine in patients with proteinui normal life span of albumin in the circulation is 15-19 days “serves the transport protein carrier for most substances > maintains oncotic pressure (or colloidal osmotic pressure) which is defined as the pressure exerted by proteins in blood plasma that tends to pull water into the circulation, thereby preventing its extravasation into the tissues si + anegative acute phase reactant, since it decreases during inflammation “high serum albumin levels are often associated with dehydration or prolonged tourniquet application or specimen evaporation ein low serum albumin levels can be related to: + inflammation + protein-calorie malnutrition = hepatic disease + burn injury + urinary loss due to kidney = edema and ascites problems "gastrointestinal loss Terms: Analbuminemia - absence of albumin : Bisalbuminemia - presence of 2 bands in the albumin region Hypoatbuminemia - decreased levels of albumin CLINICAL CHEMISTRY: “Never put off til tomorrow the book you can read today” - Holbrook Jackson 31v GLOBULINS Alpha-L-antitrypsin accounts to majority of the alpha-1 globulins Alpha-L-acid glycoprotein serves as carrier for steroid hormones Alpha-1 fetoprotein is an oncofetal tumor marker found in patients with germ cell tumor and hepatocellular carcinoma. Itis also decreased in pregnant women whose baby has Down syndrome; and elevated in pregnant women whose baby has neural tube defects Alpha-2-macroglobulin accounts to majority of the alpha-2 globulins ‘Transferrin accounts to 90% of the beta globulins ‘Transferrin is also known as siderophilin; transferrin is the transport protein for iron. Itis capable of transporting 2 ferric ions. Ferritin serves as the soluble storage form of iron Haptoglobin transports free hemoglobin; hemopexin transports heme Haptoglobin and Hemopexin decreases in patients with hemolytic anemia Ceruloplasmin is an oxidase. Itis a blue alpha-2 globulin that transports copper. 90-95% of copper in plasma is bound to ceruloplasmin, the rest to albumin. Wilson‘s disease is associated with low ceruloplasmin levels, leading to low serum copper and copper overload (deposits) in tissues Fibrinogen is the protein that is present in plasma but not in serum. It can be measured using Parfentiev method, which uses ammonium sulfate and. sodium chloride C-reactive protein serves as a non-specific indicator of inflammation. [tis found in the beta region during electrophoresis (acc. to Bishop & Kaplan; found in gamma ace. to Henry and Tietz) MISCELLANEOUS PROTEINS Bence-Jones protein are proteins identical light chains excreted in the urine of patients with MULTIPLE MYELOMA. Bence Jones Protein characteristically precipitates at 40-60°C but dissolves at 100°C. Immunofixation is the method used when to identify Bence Jones Protein. ‘Myoglobin is a heme containing protein found in the striated siletal and cardiac muscles. It can be used as marker of acute myocardial infarction. Rises at 1-3 hours Peak at 5-12 hours Returns to normal in 18-30 hours Myoglobin is not specific for AMI since it can also be elevated in muscle- related disorders. “Never put off til tomorrow the book you can read today” - Holbrook Jackson | CLINICAL CHEMISTRY:+ Troponin is a complex of three proteins: Tnl (inhibitory subunit), TaT (tropomyosin-binding subunit) and TnC (calcium-binding subunit). Cardiac isoforms of Tnl and Tn are specific for heart muscles. CardiactroponinT —_rises within 3-4 hours Peak in 10-24 hours Remains elevated for up to 10-14 days Cardiac Troponin! —_rises within 3-6 hours Peak in 14-20 hours Returns to normal in 5-10 days Il. Specimen Considerations and Patient Preparation: Serum is preferred; 24-hour urine and serous fluids can also be used Protein in CSF is LESS THAN 1% compared to PLASMA PROTEIN + Can be used to determine ifa certain body fuid is a transudate (low protein) or an exudate (high protein) ‘Noto lipemia and hemolysis lil. Methodologies of Protein Determination: £ Direct Optical Method ‘+ Absorbance of UV light at 200-225nm (or 210) and 270-290nm (or 280) have been ised to measure protein concentrations and is applied to monitor chromatographic separations of peptides and proteins « GAldahl method “+ is based on the digestion of protein with consequent measurement of nitrogen eéntent Steps: 1. Initial precipitation: serum proteins are precipitated using organic acids such as trichloroacetic acid or tungstic acid 2. acid digestion to release ammonium ions from nitrogen-containing compounds by heating with sulfuric acid in the presence of a catalyst (such as cupric sulfate; potassium sulfate; or H202 + mercury or copper), at 340-360°C. - 3. Ammonium ions that form are quantitated by alkalinization, distillation and acid titration, or by Nesslerization ° Alkalinization-Distillation-Titration is done using alkali, boric acid and HCI Nesslerization is done using Hglz and KI % Considered as the reference method; assumes average nitrogen content of proteins is, 16% (multiply N by 6.25). Actual nitrogen content of serum protein ranges from 15.1-16.8%, Some references prefer the use 6.54, which is the average nitrogen content of different proteins (albumin 6.53, alpha 6.63, beta 6.78, gamma globulins 6552, etc). The factor 6.25 is most widely accepted. % "Interferences: NPNs like urea and amino acids can cause an increase - therefore CLINICAL CHEMISTRY: precipitation testis required lever put off til tomorrow the book you can read today” - Holbrook Jackson 33 |tesa Biuret reaction \ is based on the formation of violet colored chelate between Cu?* ions and requires at least 2 peptide bonds, measured at 540nm + Biuret is a molecule that forms when urea, the end product of protein metabolism is heated at 180°C. Two molecules of urea form one molecule of biuret \ Kinetic Biuret method is preferred amino acids and dipeptides will not be detected by Biuret reaction reagents include: : a. Alkaline copper sulfate Al. Rochell b. Sodium hydroxide Sat CUSOs NaOH _KI Rochelle salt (also known as cainosimarns- | AN K used to complex cupric ion and prevent their precipitation) . Potassium iodide (keeps copper in cupric form) ‘Interferences in Biuret Reaction a. High bilirubin , Elevated lipids, Hemolyzed sample - false increased , High blood ammonia level - false decreased {igtuly (Folin-Ciocalteu) Method “= Specimen are mixed with an alkaline copper solution followed by addition of Folin- Ciocalteu reagent \ Phosphotungstomolybdic acid (Phosphotungstic acid + phosphomolybdic acid) are reduced to tungsten blue by copper complex with peptide and by tyrosine and tryptophan “Absorbance products are measured at 650-700nm; dependent on phenolic groups Dyes: Bromeresol purple ~ most sensitive, specific and precise among the dye-binding assays Bromcresol green (BCG) ~ most commonly used; measure absorbance Tetrabromphenol blue ~ used in urine reagent strip, sensitive to alburnin i Ninhydrin ~ for amino acids Methyl orange Hydroxybenzeneazobenzoic acid (HABA) Coomassie brilliant blue Pyrogallol red ~ used for analysis of fluids with lower protein concentrations such as urine and CSF CLINICAL CHEMISTRY: "Never put off til tomorrow the book you can read today” - Holbrook JacksonCHECKPOINT! Clinical C " Refractometry Rapidly estimates protein at high concentrations = Often used in urine specimen = Salt precipitation (turbidity) Test ~ used for urine and CSF a. sulfosalicylic acid ~ often used as 3% SSA, precipitates all forms of protein. b, trichloroacetic acid preferentially precipitates globulins than albumin = .sulfosalicylic acid + sodium sulfate : ._ benzethonium chloride under alkaline condition ¢. benzalkonium salts under alkaline condition. + Electrophoresis - Movement of proteins during alkaline electrophoresis, from origin towards the anode: Gamma > Beta > Alpha 2 > Alpha 1 > Albumin > Prealbumin - Gamma globulins tend to migrate towards the cathode instead of moving towards the anode during electrophoresis. This ts known as electroendosmosis. a._ First, serum is electrophoresed, at an alkaline pH (8.6). Since the pH is alkaline, proteins in serum become negatively charged, causing them to be attracted towards the positive pole or electrode known as the anode = be, After separation, regions are stained using Coomassie Brilliant blue, Amido black and Ponceau S c. ‘After staining, stained bands are quantified using densitometer a Electrophoretic patterns: > beta-gamma bridging effect - seen in patients with liver cirrhosis caused by an a increase in IgA which is found between the beta and gamma region during electrophoresis > gamma spike - seen in cases of monoclonal gammopathy (ie, multiple myeloma and Waldenstrom's macroglobulinemia) > alpha-2-macroglobulin elevation with albumin decrease - seen in patients with nephrotic syndrome alpha-1-antitrypsin deficiency - seen in patients with emphysema increased beta region - use of plasma instead of serum during electrophoresis low albumin, high alpha-1 and alpha-2 region - acute inflammation v vv IW. Values to Remember: = Reference Range: Total Protein: 65 to 8.3 g/dL. Albumin: 3.5 to 5.5 g/dL = * Globulin = Total Protein minus alburnin Conversion Factor: g/dL to g/L=10 CLINICAL CHEMISTRY: ‘tomorrow the book you can read today” — Holbrook JacksonI. Whatare Enzymes? A. Characteristics of Enzymes - Biocatalysts, mostly protein in nature, that enable the necessarymetabolic reactions to occur at body temperature, speeding up reaction rates - Act by decreasing activation energy required for the biochemical reaction to push through - Affects only the rate of the reaction; affects the rate equally on both directions (for reversible reactions); they are not used up or consumed in the reaction - Most chemical reactions in the body occur at a pH 7.0 and temperature of 37°C B. Enzymology - Definition of Terms + Holoenzyme = Whole enzyme molecule; apoenzyme + cofactor = Apoenzyme = protein portion of the enzyme = "ofactor =, ,Non-protein portion of the enzyme; can be organic or inorganic @ Coenzymes - organic (carbon-containing) cofactors Examples: __NAD/NADH or NADP/NADPH used in dehydrogenase reactions Cysteine used in creatine kinase reaction Pyridoxal (vitamin B6) phosphate used in transaminase reactions @ Activators - inorganic (non-carbon-containing) cofactors; can be metallic or non- + metallic 4 Metallic Calclum for amylase Magnesium for creatine kinase Zinc for lactate dehydrogenase Others: iron, manganese, copper“ 4 Non-metallic Chloride for amylase "= Zymogens or Proenzymes - inactive forms of enzyme Pepsinogen _- inactive form of enzyme pepsin, which promotes protein digestion ~ produced by the chief/zymogenic cells of the stomach ~ converted into pepsin upon contact with gastric acid Plasminogen _- inactive form of enzyme plasmin, which promotes fibrinolysis ~ produced by the liver ~ activated by urokinase or tissue plasminogen activator (tPA). Factors that affect enzymatic reaction Isoenzymes - enzymes that catalyze the same reaction but differ in terms of physical or chemical characteristics, tissue distribution as well as electrophoretic mobility - Example: Lactate dehydrogenase - LD1, LD2, LD3, LD4, LDS Creatine kinase - CKMM, CKMB, CKBB Alkaline phosphatase ~ intestinal ALP, placental ALP, livér ALP, bone ALP ‘Acid phosphatase - prostatic and erythrocytic ACP Macroenzymes = are high-molecular-mass forms of the serum enzymes; large enzymes - two types: macroenzyme type 1 = enzyme bound to an immunoglobulin ‘macroenzyme type 2 = enzyme bound to non-immunoglobulin substance such as drugs or plasma membrane fragments = usually found in patients who have an unexplained persistent increase of enzyme concentrations in serum ~ _ larger enzymes leads to decreased clearance of enzymes in the circulation = enzyme bound to immunoglobulin especially, have longer half-life (since IgG have lifespan of approximately 3 weeks) ~ Example: macro-Ck, macroamylase ‘Sibstrate = qeprecursor of a product in an enzymatic reaction 2° binds to the active site of the enzyme Enzymatic Reaction a ae = ee ENZYME.+ SUBSTRATE ¢> ES COMPLEX > ENZYME + PRODUCT { ee on affect the enzymes and can denature ites enzymes : = some enzymes however, perform well in. en alkaline (e.g, ALP) and acidic (eg. ACP - and pepsin) environment pH & g a = o ji o “Life isn’t about waiting for the storm to pass. It’s about learning to dance in the rain” ~ Vivian GreeneCHECKPOINT! Ci ‘Temperature ~ _ Reaction velocities of most chemical reactions increase with temperature and approximately double for each 10°C rise = most enzymes become denatured and insoluble within minutes of being subjected to temperatures of 60°C and above - Most body enzymes have temperature lncraacing ‘nayme seaety ternperatur (2C) optimum close to 37-38°C and have progressively less activity as temperature rises above 42-45°C - Low temperatures decrease enzyme activity and they may be completely inactive at temperatures of 0°C and below - _ Inactivity at low temperatures is reversible, most enzymes may be preserved by storage at -20 to -70°C Enzyme concentration - Velocity of reaction is directly proportional to enzyme concentration in the presence of excess substrate Rate of Reaction ——>> = Reaction proceeds more rapidly when more enzyme molecules are ‘Substrate concentration @ Enzyme Concentration ———2>| present to bind substrate Tie sgeoaro azcumes shat there Michaelis-Menten hypothesis ‘The higher the substrate concentration, the more substrate bound to enzyme and the greater the rate/velocity of the reaction When all enzyme is bound to substrate, there will be no further increase in velocity- this is the ‘maximum velocity When the substrate is present in an adequate amount (all enzymes are bound and saturated), the rate of ‘Enzymatie activity ‘Substrate concentration ——> reaction depends only in enzyme concentration “Life isnt about waiting for the storm to pass. It’s about learning to dance in the rain” = Vivian GreeneCHECKPOINT! C @ First order reaction - substrate readily binds to free enzyme at a low-substrate concentration - _ with the amount of enzyme exceeding the amount of substrate, the reaction rate steadily increases as more substrate is added - rate depends on substrate concentration - reaction rate is directly proportional to substrate concentration @ Zero order reaction . - high substrate concentration saturates all available enzymes; fewer binding sites" are available for attachment - reaction velocity reaches maximum; rate of increase of the velocity falls to zero - velocity is independent of substrate concentration when binding sites are saturated - rate does not depend on substrate concentration; reaction rate depends only on enzyme concentration - when product is formed, the resultant free enzyme immediately combines with excess free substrate = Inhibitors © Substances that inhil @ Competitive Inhibition ~ inhibitor binds the active site of enzyme - substrate and inhibitor compete for the same binding site enzymatic reaction ~ addition of more substrate results in the displacement of inhibitor molecules by substrate molecules , with a consequent increase in enzymatic activity ~ can be reversed by the addition of substrate molecules, @ Non Competitive Inhibition - imhibitor binds enzyme ata place other than the active site (allosteric site) of enzyme ~ alter the configuration of the enzyme in such a way as to reduce or abolish excess of the substrate to the active site, causing a decrease in enzyme activity ~ may be reversible (if binding is reversible or may be separated) or irreversible (if, inhibitor destroys a part of enzyme) ~ addition of substrate has NO effect since it cannot reverse the alteration caused by inhibitor "t about waiting for the storm to pass. It's about learning to dance in the rain Vivian Greene Seal é : gE, Importance of Enzyme Detection Greco kel © Uncompetitive Inhibition - inhibitor binds the ES complex - _ the resulting enzyme-substrate-inhibitor complex does not yield product = increasing substrate concentration results in more ES complexes to which the (=) | inhibitor binds and, thereby, increases the inhibition The Lineweave vist visi visi Competitive Uncompetitive Noncompetitive ihtbition inhibition inhibition ¥ Kyincreased Ky reduced Ky unaffected Van unaffected Vina feduced Vin reduced =) "Fnic strength or Electrolyte environment S» Blectrolytes such as calcium, magnesium, zinc and chloride are activators and may, -d by some enzymatic reactions = if onic strength is too high, enzyme activity drops + in protein-free solutions, enzymes lose activity rapidly be req = To detect tissue injury or damage = To identify abnormalities or deficiency of enzymes Measuring Enzymatic activity - Most enzyme tests used in clinical chemistry laboratory follow Michaelis-Menten kinetics and are performed under conditions of zero-order reaction ~ a large excess of substrate is present so that the amount of enzyme activity is the only rate-limiting factor in the assay - Enzymes are measured based on their activity rather than their mass-or molar concentration - Enzymatic activity = can be measured as a rate of praduct produced or as a rate of, ‘substrate consumed per unit of time + Units used in expressing enzymatic activity Enzyme activity can be expressed either in IU or KU. @ International Unit - micromole of substrate per minute; also known as U/L © Katal Unit ~ mole of substrate per second CLINICAL CHEMISTRY: “Life isn’t about waiting for the storm to pass. It's about learning to dance in the rain” = Vivian GreeneCHECKPOINT! C + Approaches to measurement of enzymatic activity: “ @ Fixed-Time (static or two-point) assay “the reactants are combined, the reaction proceeds for a de: reaction is stopped (usually by inactivating the enzyme with a weak acid) ‘measurement of a product or substance produced over a given amount of time “the reaction is assumed to be linear over the reactiod time the larger the reaction, the more enzyme is present. ®@ Multipoint Continuous Monitoring (Kinetic assays) Multiple measurements, usually of absorbance change, are made during the reaction, either at specific time intervals (usually every 30 or 60 seconds) or continuously by a continuous-recording spectrophotometer. ‘Measurement of a substrate product per minute (or hour) produced constantly over a period of time advantageous over fixed-time methods because the linearity ofthe reaction may be more adequately verified; usually preferred “ ifabsorbance is measured at intervals, several data points are necessary to increase the accuracy of linearity assessment. Continuous measurements are preferred because any devi linearity is readily observable. - selmmunoassays nowadays measure concentration of some isoenzymes (e.g. prostatic- Specific isoenzyme , CKMB mass,) - Immunoassays may overestimate active enzyme as a result of possible cross-reactivity with inactive enzymes, such as zymogens, inactive isoenzymes, macroenzymes, oF partially digestetl enzyme. = Most enzyme measurements require the use of serum since anticoagulants may: activators (like calcium and magnesium) which are necessary for enzyme reactions jon from G. Methods for Measuring Enzymatic activity + Spectrophotometric (colorimetric) ‘More cumbersome and less sensitive ‘Requires large amount of substrate (which may later on act as enzyme inhibitor) and longer incubation time + Manometry (measures pressure of gases and vapor) Evolution of gas or disappearance of gas as the reaction proceeds «= Fluorometry Coupled-enzymatic reactions “Life isn't about waiting for the storm to pass. It about learning to dance in the rain” - Vivian Greene rs a CLINICAL CHEMISTRY:erenao nuke H. Principles of Enzyme Data Interpretation 1. There is no truly “organ-specific” enzyme — since all enzymes are found in more than ones: tissue 2. Serial measurements provide most useful data; a single measurement can be misleading’) G increase or decrease) serial measurements over the course of several days checking for patterns of 3, Negative (normal) results are useful especially when ruling out presence of tissue damage 4, Enzyme date must be integrated with other information — other pertinent data must be used in conjunction with enzyme result before a diagnosis can be made 1. Classification of Enzymes 1. Oxidoreductases - oxidize one substrate and reduce the other 2. ‘Transferases - transfer a group (other than hydrogen) from one substrate to another ~ 3. Hydrolases - catalyze hydrolysis or splitting up of a bond in a substrate, resulting in the = formation of two or more molecules 4, Lyases ~ Catalyze removal of groups from substrates without hydrolysis; the product double bonds 500 ISomerases - catalyze the interconversion of geometric, optical, or positional isomers |_ Ge Aigases ~Catalyze the joining of two substrate molecules, coupled with breaking of the» Pyrophosphate bond in adenosine triphosphate (ATP) or a similar compound contai = Lactate creatine kinase acid & alkaline dehydrogenase + aspartate phosphatase * Glucose-6-phosphate aminotransferase (SGOT) _| * cholinesterase dehydrogenase, * alanine aminotransferase —_| + lipase = Malate (scPr) + trypsin dehydrogenase ‘gamma glutamy! * pepsin * Isocitrate transferase (GGT) * leucine dehydrogenase * hexokinase aminopeptidase * Cytochrome oxi + pyruvate kinase + amylase = Glutamate = glycogen phosphorylase = gluGosidase dehydrogenase + galactosidase = Beta-hydroxybutyric * 5 nucleotidase dehydrogenase = chymotrypsin + elastase glutathione-S-transferase ‘= glucose phosphate isomerase + ribose phosphate isomerase + triosephosphate isomerase * aldolase + pyruvate decarboxylase = ‘glutamate decarboxylase + tryptophan decarboxylase * glutathione synthetase CLINICAL CHEMISTRY: “Life isnt about waiting for the storm to pass. It’s about learning to dance in the rain” = Vivian GreeneIL, Enzymes Detected in the Laboratory A. Aldolase - 1. Characteristics of Aldolase - catalyzes an early step in glycolysis for glucose which ts the conversion of: anne, ae f Ald dihydryoxyacetone phosphate [it trictoce:1)diomoopirate <- car sean eas ae ome L D-glyceraldehyde-3 phosphate 2. Clinical Significance of Aldolase + Highest level in progressive muscular dystrophy ~ Elevated in skeletal muscle disease or injury, metastatic carcinoma of the liver, granulocytic leukemia, megaloblastic anemi: hemolytic anemia and tissue infarction in, general - Inmyocardial infarction: rises 6-8 hours stays elevated up to 3-4 days 3. Specimen Considerations - Noto hemolyzed sample (RBC aldolase level is 150 times as high as the serum level) ~eePlasma is preferred over serum because of the possible release of platelet enzyme during clotting 4, Methodologies 2 = sibley-Lehninger Method Substrate: fructose-1, 6-diphosphate ~ Product measured: dihydroxyacetone-phosphate + Coupled enzymatic reaction : ~ Reagent Enzymes: Triosephosphate isomerase and glycerol-3-phosphate dehydrogenase ~ Measure the decrease in NADH concentration 5. Reference Range: 2.5to 10.0U/Lat37°C Lactate Dehydrogenase (LDH) 2 1. Characteristics of LDH - - catalyzes the reversible conversion of lactate and NAD into pyruvate and NADH pans pomeeeeee ee [LACTATE + NAD* ——— - has zinc as one of its component - _ present in almost all types of tissue; tissue non-specific i'tabout waiting for the storm to pass. It’s about learning to dance in the rain™ Vivian Greene CLINICAL CHEMISTRY:2. Isoenzymes of LDH = Electrophoresis >> = Concentration in serum: LD 2>1>3>4>5 = LD4.and LDS are cold labile fractions = LD1is the most negatively charged isoenzyme - D1 is the fastest toward the anode; LDS is the slowest = Alpha-hydroxylbutryic dehydrogenase (HBD) has the same kinetic properties as LD1 but does not act on the substrate lactate - _ HBD can be measured using Rosalki-Wilkinson method, which uses ketobutyrate as a substrate with subsequent measurement of change in NADH absorbance Oren, TOTAL NORMALLY aN Bae ERG: OOS pili ete ISOENZYME eed LD; | HHAH | Heart, brain, erythrocytes | LD, _|__HHHM 3 Heart, brain, erythrocytes | “ups | HHMM | Brain, kidney | LD. | HMMM | Liver, skeletal muscle, kidney | ~ (ips [MMMM 3.( Clinical Significance ~ ‘used as a non-specific enzyme marker for acute myocardial infarction (AMD. sq Pattern of increase during AMI: Increases 12 to 24 hours after the onset of AMI Peaks at 48-72 hours after the onset of AMI Normalizes within 10 days ~ LD-flip.(.01>1,02) is oftentimes associated with AMI - Highest elevation of LDH is found in megaloblastic anemia (pernicious anemia) Liver, skeletal muscle, eum) mee ~ Conditions affecting total lactate dehydrogenase activity PRONOUNCED ELEVATION MODERATEELEVATION SLIGHT ELEVATION [J , (5 OR MORE TIMES (3-5 TIMES NORMAL) CERI Ge le Rothe) NORMAL) ik Megaloblastic anemia Myocardial infarction Most liver diseases | Widespread carcinomatosis, | Pulmonary infarction Nephrotic syndrome | especially hepatic | Hemolytic conditions Hypothyroidism | metastases Leukemias Cholangitis a | Systemic shock and hypoxia _| Infectious mononucleosis 9 | Hepatitis | Delirium tremens qd Renal infarction | Muscular dystrophy i S a o “Life isn’t about waiting for the storm to pass. Its about learning to dance in the rain” = Vivian Greenefrennons 4. Specimen Considerations and Patient Preparation + Serum is preferred, anticoagulants can inhibit LDH + No to hemolysis; LDH is a lot more concentrated in RBCs than in plasma = Do not store in freezing temperature; LDS Is cold labile, specimens for LDH should Be" | stored at room temperature 5. Methodologies: = Wacker (forward reaction) - measures enzymatic activity as lactate is converted to pyruvate S = measurement of activity can be through kinetic (uses UV) or colorimetric (addition of color reagents) ¢ KINETIC: measures the increase in absorbance at 340nm as NAD is converted to NADH 3 ¢ COLORIMETRIC: addition of phenazine methosulfate and nitroblue tetrazolium which reacts with NADH to produce a positive blue-purple color COLORIMETRIC: addition of p-nitrophenythydrazine (or 2.4 * dinitrophenylhydrazine) which reacts with pyruvate producing phenylhydrazong ~a golden brown color at alkaline pH measured at 440 or 525nm = “Wrobleuski La Due (reverse reaction) =) measures enzymatic activity as pyruvate is converted to lactate = measures the decrease in absorbance at 340nm as NADH is converted to NAD =, 3x faster than the forward reaction C. Creatine Kinase (Creatine phosphokinase) 1. Characteristics of Creatine kinase - catalyzes thé transfer of phosphate to creatine creatine + ATP <*> creatine phosphate + ADP - CK requires magnesium and thiol source (cysteine) ~ _ imbibited by zinc and manganese; excess Mg can also inhibit CK 2, Isoenzymes of Creatine Kinase = 95% of total CK activity is derived from CK-MM - Half-life of CK isoenzymes: CK-BB=2-3hours CK-MB=12hours ck-MM 5 hours, ~ Most CK are found in cytoplasm - _ mitochondrial CK is the CK found within the mitochondria of the cell. It does not appear in the circulation unless more severe cellular damage takes place “Life isn’t about waiting for the storm ta pass. Its about learning to dance in the rain” - Vivian Greene° —fellelole lle ii Ne\ei els beuiaialis| [2 ]]fs \|3 | & | a een 3. Clinical Significance of Creatine Kinase - CK-MB in AME: rises after 4 to 8 hours peaks at 12-24 hours returns to normal within 48 to 72 hours (2-3 days) ¢ - Highest elevation of creatine kinase is found in Duchenne’s muscular dystrophy \ - NOT present in liver and in RBCs CONDITIONS AFFECTING CREATINE KINASE POUT PTO eT ee OREN NON) (2-4 TIMES NORMAL) Duchenne’s muscular Severe exercise, trauma, surgical i dystrophy procedure, intramuscular injections “ Polymyositis Delirium tremens, alcoholic myopathy Dermatomyositis severe ischemic injury Myocardial infarction Pulmonary infarction Pulmonary edema (some patients) Hypothyroidism Acute agitated psychoses 4: “Specimen Considerations and Patient Preparation: | Noto hemolysis. CK is NOT present in RBCs. However, adenylate kinase, which . catalyze a similar reaction as that of CK is present, causing a false increase in CK when hemolysis is present + Stored in the dark since it has been found that CK is inactivated by light = CK levels are usually higher in males than in females due to differences in muscle mass. = _ Inactivation of CK can partially be reversed by addition of sulfhydryl compounds ({.e. N-acetyleysteine, mercaptoethanol, thioglycerol, and dithiothreitrol) to the assay reagent 5. Methodologies - Sulfhydryl activator (e.g, cysteine, N-acetyl cysteine, glutathioné, dithiothreitol or Cleland’s reagent, monothiolgycerol) is required - Methods: + Tanzer-Gilvarg (forward reaction) ATP + creatine > ADP + creatine phosphate ADP formed from the forward CK reaction of CK is reacted with PK and LDH. Pyruvate kinase: ADP + phosphoenol pyruvate ¢ ATP + pyruvate LDH: pyruvate + NADH © lactate + NAD* *SNADH absorbs light at 240nm; NAD does not; pH is maintained at 9.0 CLINICAL CHEMISTRY: “Life isn’t about waiting for the storm to pass. It's about learning to dance in the rain” = Vivian GreeneCHECKPOINT + Oliver-Rosalkd (reverse reaction) ADP + creatine phosphate > ATP + creatine ATP formed from the reverse CK reaction is reacted with HK and G6PD ‘Hexokinase: ATP + glucose © ADP + glucose-6-phosphate GGED: G6P + NADP* + 6-phosphogluconate + NADPH “NADPH absorbs light at 340nm; NADP does not “+pil is maintained at 6.7 Reverse reaction is 6X faster compared to forward reaction = CK-MB mass vs CK-MB - CK-MB mass assay uses monoclonal antibody technology ~ it does not depend on protein’s enzymatic activity unlike the usual CK-MB assays D. Aspartate Aminotransferase/ Transaminase (AST) a 1. Characteristics of AST - previously known as the serum glutamtc-oxaloacetic transaminase (SGOT) - catalyzes a reversible reaction between: ASPARTATE + a-KETOGLUTARATE + OXALOACETATE + GLUTAMATE © requires pyridoxal phosphate as coenzyme =) widely distributed in tissue; highest in cardiac tissue, liver, muscle 2: Clinical Significance of AST =» often used in conjunction with ALT for hepatocellular disorders = AST during AMI: rises after 6-8 hours peaks at 24 hours returns to normal within 5 days + conditions affecting AST ON OL (5 ORMOREXN) (ena) ea eeay) ‘Acute hepatocellular damage | Biliary tract obstruction _| Pericarditis Myocardial infarction Cardiac arrhythmias Circhosis = Circulatory collapse (shock) | Congestive heart failure _| Pufinonary infarction “ Acute pancreatitis, | Metastatic or primary Delirium tremens Infectious mononucleosis tumor in liver Cerebrovascular | Muscular dystrophy accident 3. Specimen Considerations - Hemolysis should be avoided because it can dramatically increase serum AST concentration. > AST activity is stable in serum for 3 to 4 days at refrigerated temperatures CLINICAL CHEMISTRY: “Life isn't about waiting for the storm to pass. It's about learning to dance in the rain” ~ Vivian Greenefteccront 4, Methodologies + Karmen method: coupled enzymatic reaction + Enzymatic reaction for AST: aspartate + alpha ketoglutarate + oxaloacetate + glutamate - Add malate dehydrogenase: oxaloacetate + NADH © malate + NAD* Measure loss of absorbance at 340nm due to formation of NAD* Optimal pH is 73-78 + Diazonium salt formation of diazonium derivative = Colorimetric method: Reitman-Frankel reagent: dinitrophenylhydrazone - formation of blue color, measured at 505nm - lack of specificity, reacts with any keto-compound . Alanine Aminotransferase/Transaminase (ALT) 1. Characteristics of ALT © previously known as serum glutamic-pyruvic transaminase (SGPT) my catalyzes: ALANINE + a:KETOGLUTARATE + GLUTAMATE + PYRUVATE 2x. Clinical Significance = Highest elevation is found in acute liver hepatitis, - specific forliver disease compared to AST 3, Specimen Considerations = ALTis stable for 3 to 4 days at 4°C + relatively unaffected by hemolysis. 4, Methodologies Enzymatic method - Enzymatic reaction for ALT: alanine + alpha ketoglutarate © pyruvate + glutamate + Add lactate dehydrogenase: pyruvate + NADH © lactate + NAD* Measure loss of absorbance at 340nm due to formation of NAD* Optimal pH is 73-7.8 <* Diazor um salt formation of diazonium derivative “Life isn't about waiting Jor the storm to pass. I's about learning to dance in the rain” ~ Vivian Greene CLINICAL CHEMISTRY:"Colorimetric method: Reitman-Frankel initrophenylhydrazone - formation of blue color, measured at S0Snm_ + lack of specificity, reacts with any keto-compound F, Alkaline phosphatase 1. Characteristics of ALP . - Catalyzes hydrolysis of phosphomonoesters (or organic phosphate esters) into alcohdt and phosphate at an alkaline pH (9.0-10.0) - Requires activator zine 2, Isoenzymes - Normal isoenzymes: intestinal, placental, bone and liver ~ Liver and bone ALP are the most predominant fractions - canbe differentiated using electrophoresis, heat denaturation and chemical inhibition - Carcinoplacental isoenzymes include Regan, Nagao and Kasahara. They are usually found in patients with malignancy and their characteristics resemble that of placental ALP *" Electrophoresis (origin towards anode) INTESTINAL > PLACENTAL > BONE > LIVER ~ Liver and bone fractions are difficult to resolve during electrophoresis + Toimprove separation of bone and liver forms, use: @ neuraminidase (to remove sialic acid) ®@ wheat germ lectin (to bind other isoenzymes) = Heat denaturation (most heat stable to most heat labile) > PLACENTAL > INTESTINAL > LIVER > BONE - Heat stability is determined by heating serum at 56°C for 10-15 minutes Placental ALP ~ most heat stable of all the normal ALP isoenzymes (up to 60°C for 10 mins) Regan ALP ~ most heat stable among all the types of ALP + Chemical Inhibition L-phenylalanine ~ inhibits placental, intestinal, Regan and Nagao Levamisol ~ inhibits liver and bone isoenzymes L-homoarginine ~ inhibits liver and bone isoenzymes 2M Urea ~ inhibits bone isoenzyme Leleucine ~ inhibits Nagao isoenzyme 20% ethanol ~ denatures liver ALP rapidly than bone e©e@eeee : “Life isn’t about waiting for the storm to pass. IP's about learning to dance in the rain” ~ Vivian Greenetieccconukenne 3, Specimen Considerations “absorbance is measured at 400-410nm \ original BLB method uses glycine “= buffer Bowers-MeComb | p-Nitrophenyi |“ Uses phosphate-accepting buffer (a ©] (comb) phosphate transphosphorylating buffer, i. AMP - No hemolysis since ALP is 6x more concentrated in RBCs than serum ~ ALP assays should be run ASAP after collection since activity in serum increases approximately 3% to 10% on standing at 25°C or 4°C for several hours ay - Diet may induce elevations in ALP activity of blood group B and 0 individuals who are secretors. - Values maybe 25% higher following ingestion of a high-fat teal due to increase Intestinal fraction 4, Methodologies for Total ALP Activity rar COUNTS Shinowara-jones- | Beta- ‘Long incubation time Reinhart | glycerophosphate_|“+_high blank values King-Armstrong | Phenylphosphate | = Endpoint, requires protein removal Bessey-Lowry- | p-Nitrophenyl__ |= Endpoint or kinetic, rapid Brock phosphate | = p-nitrophenol (colorless end product) atalkaline pH forms quinoid structure with intense yellow color = “reaction time is 30 minutes; reaction is stopped using NaOH (inactivates enzyme and acts as color developer) buffer also known as 2-amino-2- methyl-1-propanolol) at pH 10.5 at 30°C “Other modifications include use of diethanolamine as buffer | “reference method “+ measures p-nitrophenol (yellow) | characterized by increased absorbance oe ee | Sat 405m = | #nitrophenyt |“ “Kinetic; automated + | phosphate Measurement of ¢-nitrophenoxide | Eick dine si overt ot ee CLINICAL CHEMISTRY: “Life isn't about waiting for the storm to pass. Its about learning to dance in the rain™ = Vivian Greene- _ Often used in evaluation of hepatobiliary (obstructive conditions) and bone disorders, ~ (osteoblast involvement) & + Highest elevation of ALP is seen in: Paget's disease (osteitis deformans) &S : hyperparathyroidism, rickets and osteomalacia, fractures, and malignant = ~ Increased i tumors - Hepatocellular disorders (cirrhosis and hepatitis) producesonly slight elevations (les, than 3.x ULN) = fA ~ Biliary obstruction (cholestasis) ~ 3-10 times elevation - Physiologic elevation of ALP can be seen in growing children due to osteoblastic activity = Conditions affecting alkaline phosphatase PRONOUNCED ELEVATION MODERATEELEVATION SLIGHT ELEVATION (ecko sanrel tan) (ema) (UPTO3xN) Bile duct obstruction " Granulomatous or Viral hepatitis (intrahepatic or extrahepatic) infiltrative diseases of _| Cirrhosis ry cirthosis liver Healing fractures itis deformans (Paget’s _ Infectious mononucleosis_| Pregnancy (® ase) | Metastatictumorsin bone | placental ALP) | Metabolic bone diseases | Normal growth (oi patterns in children kets, osteomalacia G. Acid Phosphatase ‘ES Characteristics of Acid Phosphatase = Catalyzes hydrolysis of phosphomonoesters (or organic phosphate esters) into alcohol and phosphate at an acid pH (5.0-6.0) - Greatest concentrations occur in prostate, iver, spleen, RBCs, platelets and bone - Highest concentrations in prostate gland secretions and erythrocytes sha - Lysosomal, prostatic, erythrocyte, macrophage, and osteoclastic ACPs are five Important types found in humans - Tartrate-resistant acid phosphatase can be found in hairy cell leukemia 2. Isoenzymes "Electrophoretic separation Erythrocytic ACP remains in origin Prostatic ACP migrates with great mobility = Chemical Inhibition: ~ inhibited by 2% formaldehyde solution _| ~ inhibited by L-tartrate and ImM cupric sulfate solution ~ specific acid phosphatase ~ non specific acid phosphatase CLINICAL CHEMISTRY: “Life isn’t about waiting for the storm to pass. It's about learning to dance in the rain” ~ Vivian Greenerecnsonts eae 3. Specimen Considerations = Specimen must be acidified to prevent loss of ACP. Citrate is the preferred anticoagulant, buffered to a pH of 62-66 - Plasma is the sample of choice to minimize platelet contamination ~ Serum should be separated from clot to prevent leakage of ACP from RBCs and platelets ~ Serum activity decreases within 1 to 2 hours at room temperature without the addition of a preservative, due to loss of carbon dioxide in the sample which results in an inerease in pH ~ fot assayed immediately, serum should be frozen or acidified to a pH lower than 6.5 = Hemolysis should be avoided because of contamination from erythrocyte ACP, causing false elevation. ~ Fluoride inhibit ACP; oxalate and heparin causes a false decrease 4, Methodologies for Total ACP Activity COO Se co ‘Bodansky Beta-glycerophosphate | Lengthy assay, nonspecific jutman, King Phenylphosphate ‘Nonspecific Armstrong _ pss pier: ae ‘Hudson Pp Rapid, nonspe } nitrophenylphosphate | Substrate is converted to p- | nitrophenol, addition of a base | produces p-nitrophenolate (yellow) which is measured at 410nm Babson and Reed | Alpha- ‘Complicated, less sensitive; preferred | ______| naphthylphosphate __/ for continuous monitoring reactions _| Roy Thymolphthalem ‘more specific for prostatic form; | : monophosphate | method of choice for quantitative | | endpoint reactions ‘measurement of product | seu | oe ae thymolphthalein at 590nm | Rietz, Guilbault | 4Methylumbelliferone | Fluorescent, some improved | phosphate nsitivity | 5. Clinical Significance Elevated in patients with prostatic carcinoma, however itis not specific since it can also be elevated in prostatic hyperplasia and prostatic surgery Useful in forensic clinical chemistry ~ especially in medico legal evaluation of rape; ACP can be detected in vaginal washings for up to 4 days; it can also be detected in beddings, undergarments or clothes stained with seminal fluid in the crime scene ACP may also be elevated in bone diseases due to osteoclastic activity, as well as in Gaucher disease “Life isn't about waiting for the storm to pass. Its about learning to dance in the rain” ~ Vivian Greene