MTAP-1-CLIN-CHEM-NPNs 2.pdf

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | NON-PROTEIN NITROGENOUS SUBSTANCES | MTAP 1 MIDTERMS ____________________________________________________________________________________...

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | NON-PROTEIN NITROGENOUS SUBSTANCES | MTAP 1 MIDTERMS ____________________________________________________________________________________ OUTLINE UREA I. Non-Protein Nitrogenous Major end product of PROTEIN § Substances metabolism § Clinically Significant NPNs Ø 90% excreted § Disease Correlations Ø 10% reabsorbed II. Renal/Kidney Function Tests § First to INCREASE in a renal disease § Laboratory Methodologies § Synthesized solely in the liver from § Precautions and Specimen CO2 and ammonia (urea/Kreb's Considerations henseleit cycle). § Reference Ranges § Concentration in the plasma is an indicator of renal function, dietary § NON-PROTEIN NITROGENOUS intake of proteins and level of SUBSTANCES protein metabolism. § Toxic by-product which is normally § Substances in the blood which contain removed from the blood by the nitrogen but are not considered as kidneys. proteins § If the kidneys are impaired, § End products of the metabolism of urea is not removed in the nucleic acids, amino acids and proteins blood and accumulates in The test for NPN’s (specifically UREA the blood. and CREATININE) is considered a § An increased concentration of KIDNEY FUNCTION TEST (KFT) serum/plasma urea may indicate a KIDNEYS flaw in the filtering system of the kidneys. § Paired, bean-shaped organ located retroperitoneally § measure by its nitrogen content (Urea = BUN x 2.14) § Each kidney has 1.5 million nephrons - the functional unit of the kidney § Screening procedure for kidney function § Receives approximately 20-25% of the total cardiac output § 70-80% glomerular damage before the concentration of urea be § Functions: increased in the blood. o Elimination of waste products § Major ORGANIC constituent of urine o Maintenance of blood Volume o Easily removed by dialysis o Maintenance of electrolyte balance § Urea results are effective for diagnosis if combined with o Maintenance of acid-base creatinine results balance AZOTEMIA o Endocrine function (EPO § elevated level of nitrogenous secretion) substances (NPN’s) like urea and creatinine in the blood. § CLINICALLY SIGNIFICANT NPNs Ø Pre-renal azotemia § caused by reduced renal blood Plasma Concentration (% flow NPN Compounds of Total NPN) § Associated with the following: UAUCCA o high protein diet Urea 45-50% o congestive Heart Failure Amino Acids 20% o shock Uric Acid 10% Creatinine 5% o hemorrhage Creatinine 1-2% o increased protein Ammonia 0.2 catabolism  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | NON-PROTEIN NITROGENOUS SUBSTANCES | MTAP 1 MIDTERMS ____________________________________________________________________________________ o corticosteroid therapy anemia (normocytic, normochromic) Ø Renal azotemia uremic frost (dirty skin) § caused by damage within the generalized edema kidneys foul-breath § Associated with damage within sweat is urine-like the kidneys § responsible for the changes in RBC § Acute & Chronic renal failure, shape (burr cells) glomerulonephritis Ø Post-renal azotemia CREATININE § urinary tract obstruction § Major end product of MUSCLE § Renal stones metabolism § inflammatory conditions § 100% excreted; § tumor or malignant growths o maximum of 1% reabsorbed § Creatinine is directly proportional to UREA:CREA RATIO muscle mass. Normal Urea: Crea Ratio CHEA : No Dont o Partially secreted by the PCT 20 : 1/10 : / o Not affected by protein diet Ø 10:1 / 20:1 Pre-renal azotemia o Not easily removed by dialysis § Increased plasma urea § Can be used to: § Normal plasma crea o check for the completion of a 24-hour urine § Increased Urea: Crea Ratio o evaluate fetal maturity Post-renal azotemia § as gestation progresses, more creatinine is # Ø Increased plasma urea excreted by the fetus Ø Increased plasma crea into the amniotic fluid - Ø Increased Urea: Crea Ratio (2mg/dL) § THE BEST INDEX OF KIDNEY FUNCTION Decreased Urea:Crea Ratio § not reused by the body (solely a Ø decreased urea production waste product) UREA DISEASE CORRELATIONS § Reflects the capability of the kidney to excrete waste material o Urea levels in the blood are DECREASED in the following conditions: § Not elevated unless 25-50% of the nephrons are destroyed § Poor nutrition § High fluid intake BLOOD URIC ACID (BUA)/URATE § Excessive intravenous (IV) fluids Major end product of PURINE and/or NUCLEIC ACID metabolism § Pregnancy § Severe liver diseases Formed from xanthine by the action of xanthine oxidase in the liver and § Effects of some hormones intestine. § Severe vomiting and diarrhea At a pH of 7.4 ; more than 95% of uric acid in the body fluids exist as monosodium o UREMIA urate § a clinical syndrome comprised of a o Two major sources: marked ELEVATION in plasma urea § Endogenous- nucleic accompanied by acidemia and acid metabolism electrolyte imbalance. § Exogenous- uric acid in § kidneys fail to eliminate waste the diet (vegetables and legumes) products o 90% reabsorbed &10% excreted o characterized by:  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | NON-PROTEIN NITROGENOUS SUBSTANCES | MTAP 1 MIDTERMS ____________________________________________________________________________________ KIDNEY FUNCTION TESTS URATE DISEASE CORRELATIONS Involves all testing procedures that can § Hyperuricemia assess the status of the kidneys. o increased uric acid It is divided into three groups mainly: concentration in the blood (> 7 § Glomerular Filtration Tests mg/dL) § Tubular Reabsorption Tests o Causes: § Tubular Secretion Tests § Increased uric acid production UREA § Decreased renal 1. Micro-Kjeldahl (INDIRECT) excretion CLASSICAL REFERENCE METHOD § § Increased Uric Acid Production: FOR UREA a. Digest Nitrogen to NH3 (Kjeldahl o Increased nuclear metabolism Process) (Lymphomas, leukemia, multiple myeloma etc.) § Uses an acid digestion mixture o Ingestion of certain drugs § Nitrogen ammonia (NH3) o Excessive consumption of purine- b. Measurement of ammonia rich foods Ø Micro-Kjeldahl Nessler o Obesity § Nessler’s Reaction (Nesslerization) o Hypertriglyceridemia § dimercuric potassium iodide (Nessler’s reagent) § Decreased Uric Acid Excretion: o Hypertension Ø Micro-Kjeldahl Berthelot o Chronic Renal Failure § Berthelot Reaction o Ketoacidosis § Gout o degenerative disorder; commonly in males o deposition of uric acid crystals in the joints (tophi) 2. DAM: Diacetyl Monoxime (DIRECT) § EMPLOYED IN AUTOANALYZER § LESCH-NYHAN SYNDROME o inborn errors of metabolism o deficiency of hypoxanthine § Reaction is intensified by: guanine phosphoribosyl § ferric ions and thiosemicarbazide transferase (HGPRT) needed for à intense red color formed is measured at 540 nm recycling of degraded nucleotides § ADVANTAGES: o Simple § Clue: finding orange-sand in diapers o Direct measurement of UREA § Complication: can lead to o Shows no interference by mental retardation ammonia 3. Enzymatic Method (Indirect) § Treatment: Allopurinol a. Urease-Nessler AMMONIA § Major end product of AMINO ACID metabolism § Synthesized solely in the liver by deamination of amino acids b. Urease-Berthelot § it is NOT a good indicator of kidney function. § Mostly increased in severe liver diseases, hepatico coma or in Reye’s syndrome. § PART OF THE LIVER FUNCTION TESTS  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | NON-PROTEIN NITROGENOUS SUBSTANCES | MTAP 1 MIDTERMS ____________________________________________________________________________________ c. COUPLE DEHYDROGENASE/GLUTAMATE CREATININE DEHYDROGENASE (GLDH) METHOD 1. Direct JAFFE Reaction Method (1886) Candidate Reference Method § Creatinine in protein free-filtrate is reacted with alkaline picrate to form a colored complex Creatinine (serum) alkaline↓picrate red-orange tautomer of creatinine picrate § Reagent: ALKALINE PICRATE o picric acid (trinitrophenol) 4. ISOTOPE DILUTION/MASS SPECTROMETRY dissolved in 10% NaOH (IDMS) Gold Standard o should be freshly prepared Reference method due to its because prolonged standing will high cost convert picric acid to picramic acid PRECAUTIONS, SPECIMEN CONSIDERATIONS § Disadvantages: § Specimens: Plasma, Serum or Urine o Not specific for creatinine § NONHEMOLYZED sample is o Affected by interferences; recommended § False Increased o Highly affected by protein diet Ketones o BUT the effect of a recent protein meal Glucose is minimal # KIDS GO ZOR PIZZA Fructose ↓ Usually Afte Mrs , § fasting sample is NOT required usually Protein § Fluoride and citrate are inhibitors of UREASE enzyme Urea § Avoid contamination with ammonium Ascorbic acid salts à can become ammonia Cephalosporins § Avoid prolonged standing of sample § False Negative àbecause urea will be converted to Bilirubin ↓ BH ammonia Hemoglobin § Urea is susceptible to bacterial decomposition § To remove interferences use the following as these two reagents eliminate the § Specimens (particularly urine and timed interferences making the method both urine samples) that cannot be analyzed specific and sensitive: within a few hours should be refrigerated. o Method of Hare (Lloyd’s Reagent) UREA REFERENCE RANGE § Sodium aluminum silicate BUN: o Fuller’s Earth Reagent Conventional unit: § Aluminum Magnesium 8 – 23 mg/dL silicate SI unit 2. Kinetic Jaffe Method 2.9 – 8.2 mmol/L Differential rate of color Conversion Factor: development of non-creatinine Conventional to SI unit à 0.357 chromogens is employed thus Remember! eliminating interferences Values are lower in children Popular, inexpensive, rapid and Males have slightly higher values easy to perform compared to females Requires automated equipments PFF is not needed, SERUM is used directly  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | NON-PROTEIN NITROGENOUS SUBSTANCES | MTAP 1 MIDTERMS ____________________________________________________________________________________ 3. Enzymatic Method Conversion Factor: No interference from glucose and other Conventional to SI unit à 88.40 Jaffe chromogens Remember! a. Creatinine Iminohydrolase Method Values are lower in children Males have slightly higher values compared to females URIC ACID 1. Cyanide Method (REDOX) (Folin, Brown, Newton, Benedicts) b. Creatinine Aminohydrolase Method o Phosphotungstic acid has reducing property via Jode o NaCN is the color stabilizer; toxic; not readily available 2. Sodium Carbonate Method (Archibald, Henry, Caraway) 4. Isotope Dilution/Mass Spectrometry o Sodium carbonate is not toxic (IDMS) 3. Enzymatic Method ULTIMATE REFERENCE STANDARD for o Uricase Method creatinine measurement. § SIMPLEST AND MOST SPECIFIC METHOD PRECAUTIONS, SPECIMEN CONSIDERATIONS § CANDIDATE REFERENCE METHOD for uric acid Specimens: Plasma, Serum, or Urine § Uric acid has a UV absorbance peak at 293 Hemolyzed samples should be avoided nm; Allantoin DOES NOT have o considerable amounts of non- a UV peak at that creatinine chromogens are wavelength present in RBC’s. § Upon addition of uricase, ALLANTOIN will be Lipemic and icteric samples produce formed. erroneous results. § Decrease in absorbance If stored at proper conditions it must be = to the concentration of uric acid maintained at a pH of 7.0 Fasting sample is NOT required o although high-protein ingestion may transiently elevate serum concentrations. Urine should be refrigerated after collection 4. Isotope Dilution/Mass Spectrometry Urine should be frozen if longer storage § The sample is diluted with known than 4 days is required. amount of isotope. Drugs may affect results § The CANDIDATE REFERENCE METHOD by the National Institute o Dopamine of Standards o Lidocaine CREATININE REFERENCE RANGE Serum or plasma CREATININE Adult: 0.6–1.2 mg/dL (53–106 μmol/L) Children 126 mg/dL on more § CRITERIA FOR DIAGNOSIS: than one testing o Fasting plasma glucose ≥126 § Clinical findings: mg/dL (7.0 mmol/L) on at least ↑ plasma and urinary glucose two occasions ↑ urine specific gravity o Random plasma glucose level ↑ serum osmolality (or 2 hours post glucose load level) ≥200 mg/dL (11.1 mmol/L) ↓ blood and urine pH (acidosis) o Glycated hemoglobin (HbA1c) ketonemia and ketonuria ≥6.5% on at least two occasions + + electrolyte imbalance (↓Na, ↑K , - ↓HCO3 ) CLASSIFICATION OF DIABETES MELLITUS ↓ pCO2 due to kussmaul kien A. TYPE 1 DIABETES respirations § former names: Ø Insulin-dependent diabetes mellitus Ø juvenile onset diabetes mellitus Ø Brittle diabetes Ø Ketosis-prone diabetes § due to autoimmune (Tc) destruction of the beta cells of the pancreas (Type IV hypersensitivity) § responsible antibodies: Ø Insulin autoantibodies (IAA) 2. HYPOGLYCEMIA Ø Glutamic acid § involves decreased glucose decarboxylase autoantibody levels and has many causes (GAD65)  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ Ø Islet cell autoantibodies (ICA512) § Diabetic individuals have insulinopenia due to loss of pancreatic beta-cells and thus requires lifetime administration of insulin to prevent onset of ketosis § Associated with HLA-DR3 and HLA-DR4 (encoded by MHC Class II genes on p- arm chr.6) § Signs and symtoms: TYPE 1 / LATE TYOE EARLY TYPEL 2 o Polyuria N ↑ INULLY ↓ Nour C- PEP N C-PEP o Polyphagia o Polydipsia o rapid weight loss OTHER SPECIFIC TYPES OF DIABETES o hyperventilation 1. Pancreatic disorders /pancreatectomy o mental confusion 2. Endocrine disorders: Cushing's syndrome, o possible loss of consciousness. phaeochromocytoma, acromegaly, hyperthyroidism § Complications: 3. Drug or chemical inducers: dilantin and o Nephropathy pentamidine (anticonvulsants) 4. Genetic syndromes: Down syndrome, o neuropathy and retinopathy Klinefelter syndrome, Turner syndrome o nontraumatic amputations etc. o DIABETIC KETOACIDOSIS 5. Exocrine disorders: cystic fibrosis, neoplasia, hemochromatosis (may lead B. TYPE II DIABETES to BRONZE DIABETES) § former names: § GESTATIONAL DIABETES MELLITUS o non-insulin dependent DM § Inability to metabolize carbohydrates o adult-type/maturity-onset DM usually due to hormonal changes o stable diabetes occurring in pregnancy and o ketosis-resistant disappearing after delivery. o receptor-deficient DM § hyperglycemia due to insulin resistance § Glucose intolerance that develops and defective insulin secretion during approximately 7% of all § It has milder symptoms as compared to pregnancies type I DM, however; § Screening should be performed between o untreated type II DM will result to 24-28 weeks of gestation hyperosmolar hyperglycemic state (HHS) due to accumulation § GDM converts to DM within 10 years in 30- of glucose accompanied by: 40% of cases § severe dehydration § Diagnosis: § electrolyte imbalance (low Na, high K) o one-step approach (OGTT): for § increased BUN and high-risk women INCIN DEPENDENT creatinine. & ~ NON-nm o two-step approach (initial screening test followed by OGTT): women with average risk OGTT REQUIREMENTS 1. Patient should be ambulatory (mobile). § CHO depletion and inactivity or bed rest impair glucose tolerance  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ 2. The test should be performed after an overnight Ø 8 to14-hour fasting (not longer than 16 hours) Ø Test is done only in the morning 25 3. Unrestricted diet of 150g carbohydrate/day for 3 days prior to 15 testing 4. Individual should not eat food, drink tea, coffee, or alcohol, or smoke cigarettes INTRAVENOUS GLUCOSE TOLERANCE TEST during the test, and should be seated. § Used for diabetics with gastrointestinal 5. Must be done at 24-28 weeks of (GIT) disorders gestation. § 0.5 g of glucose/kg body weight ONE STEP METHOD (given within 3 minutes) is administered INTRAVENOUSLY Ø IADPSG Consensus § Collect blood after 5 minutes of IV 1. Collect fasting plasma (8-hour). glucose administration 2. Give 75 gram glucose § Indications for the use of IVGTT: 3. Collect plasma after 1 and 2 hr. o Unable to tolerate large carbohydrate in the diet Ø GDM is diagnosed if any of the following plasma glucose values are exceeded: o Presence of altered gastric physiology § Fasting: ≥92 mg/dL (5.1 mmol/L) o Previous operation or surgery of § 1 h: ≥180 mg/dL (10.0 mmol/L) the gastrointestinal tract § 2 h: ≥ 153 mg/dL (8.5 mmol/L) o Presence of chronic malabsorption syndrome TWO STEP METHOD VI. LABORATORY: CHO Ø NIH Concensus Specimens for carbohydrate analysis: v STEP 1 ü Whole blood 1. Give 50 gram glucose to a ü Serous fluid nonfasting patient. ü Plasma 2. Collect plasma after 1 hr ü Synovial fluid 3. If the plasma glucose level is >140 ü Serum mg/dl, proceed to Step 2. If not, STOP. ü CSF v STEP 2 ü Urine 4. Collect fasting plasma (8 hour). Standard clinical specimen: FASTING VENOUS PLASMA 5. Give 100 gram glucose. SPECIMEN CONSIDERATIONS 6. Collect plasma after 1, 2, and 3 hr. Serum is appropriate for glucose analysis 7. GDM is diagnosed if any of the if it is separated from the cells following plasma glucose values are immediately after centrifugation exceeded: (approximately 30 minutes after blood collection) o Bacteria, WBCs and RBCs à can LOWER glucose results  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ If processing will be delayed for GLUCOSE METHODOLOGIES more than 30 minutes, use SODIUM I. CHEMICAL METHODS (oxidation- FLUORIDE reduction methods) o Flouride binds magnesium, which 1. Copper reduction inhibits the enzyme ENOLASE necessary for glycosis. 1.1 Folin Wu Method § Causes false negative 1.2 Nelson Somogyi BUN result. (Fluoride 1.3 Neocuproine method inhibits urease!) 1.4 Benedict’s method If whole blood is refrigerated, 2 mg of NaF/mL of whole blood prevents 1.5 Clinitest Method glycolysis for up to 48 hours 2. Ferric reduction (Hagedorn- Fasting blood sugar should be obtained Jensen) after 8-10 hours of fasting 3. Condensation method (Ortho- CSF glucose concentration is Toluidine) approximately 60-70% that of plasma II. ENZYMATIC METHODS concentrations. 1. Glucose Oxidase Blood glucose should be obtained 1-2 hours BEFORE spinal tap. 2. Hexokinase-G6PD Peritoneal fluid glucose is the same as III. ISOTOPE DILUTION GAS plasma glucose CHROMATOGRAPHY/MASS SPECTROMETRY Whole blood gives approximately I. CHEMICAL METHODS 10-15% LOWER glucose levels than serum or plasma. 1. COPPER REDUCTION METHOD Venous blood glucose is 7mg/dL LOWER o OLDEST METHOD than capillary blood glucose due to § Principle: Glucose and other tissue metabolism reducing sugars convert cupric to Capillary blood glucose is the same with cuprous ions in the presence of heat arterial blood glucose. and alkali Glucose is metabolized at room temperature at a rate of 7mg/dl/hr. At 4°C, glucose decreases by approximately 2mg/dl/hr. The rate of metabolism is higher with bacterial contamination or leukocytosis. In serum specimens without bacterial a. FOLIN WU METHOD contamination or leukocytosis, results are o SENSITIVE but not specific clinically acceptable for up to 90 minutes before separation of serum from cells. o MAJOR DISADVANTAGE: non-glucose reducing Direct methods for measuring β- substance also reacts with hydroxybutyrate are now replacing the the tests strips and tablets for urine ketone testing. Because β-hydroxybutyrate levels are high in diabetic ketoacidosis (DKA) and fall with treatment, whereas acetoacetic acid and acetone levels rise with treatment, urinary ketone strips are not useful for monitoring therapy. Calculation of the anion gap is employed instead to monitor recovery from DKA.  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ ferrocyanide by reducing sugars b. NELSON SOMOGYI METHOD Disappearance of color is o SENSITIVE and SPECIFIC measured o After PFF preparation, non- at 400 nm glucose reducing substances are adsorbed by barium Employed in AUTOANALYZERS sulfate c. NEOCUPROINE METHOD 3. CONDENSATION METHOD § Principle: The aldehyde group of glucose condenses with aromatic amines in hot acetic acid solution to form colored derivatives d. BENEDICT’S METHOD § Modification of Folin Wu § ORTHO-TOLUIDINE METHOD § Used to detect and quantify § a.k.a. DUBOWSKI METHOD reducing substances in body fluids § MOST SPECIFIC NON- § Uses CITRATE and TARTRATE as ENZYMATIC METHOD for stabilizing agent glucose measurement § Negative result: BLUE § MAJOR DISADVANTAGES: § Positive result: carcinogenic and GREENàYELLOWàBRICK RED teratogenic PPT e. CLINITEST TABLET § Detects ALL reducing sugars § uses URINE as sample § Watch out for “pass through” phenomenon o occurs when glucose II. ENZYMATIC METHODS level is VERY HIGH o Remedy: use TWO Measures only GLUCOSE and not other instead of five drops reducing sugars Three enzyme systems are commonly used to measure glucose: 1. Glucose Dehydrogenase 2. Glucose Oxidase a. Colorimetric b. Polarographic 3. Hexokinase NOTES TO REMEMBER 2. FERRIC REDUCTION o Glucose exists either as alpha- a.k.a. HAGEDORN JENSEN METHOD glucose or beta-glucose. Principle: INVERSE COLORIMETRY § Alpha-glucose = 35% o Reduction of yellow § Beta-glucose = 65% ferricyanide to colorless  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ o Alpha glucose is converted into FALSELY DECREASED beta glucose using the enzyme results mutarotase. o Presence of bleach and 1. GLUCOSE DEHYDROGENASE METHOD detergents, can cause FALSELY INCREASED § Glucose is measured results spectrophotometrically or via a change in electrical current § Polarographic Glucose Oxidase Method A. Spectrophotometric o Oxygen depletion is measured and is proportional to the 2. GLUCOSE OXIDASE METHOD amount of glucose present. § Most specific enzyme reacting with only ß -D-glucose o H2O2 is prevented from re-forming O2 by adding molybdate, iodide, catalase and ethanol NOTE: o Glucose oxidase is not the reference method § Colorimetric Glucose Oxidase Method § Glucose oxidase is very specific, it only measures beta-glucose. § Glucose oxidase is affected by reducing and oxidizing agents. 3. HEXOKINASE METHOD Ø REFERENCE METHOD/ GOLD STANDARD TEST Ø MOST COMMONLY USED METHOD to o a.k.a. Saifer determine serum glucose levels Gernstenfield Method Ø Measures both alpha & beta D-glucose o The coupled reaction involved in glucose Ø Glucose concentration is proportional to oxidase method is known the rate of production of nicotinamide as TRINDER’S REACTION adenine dinucleotide phosphate (NADPH) o High concentrations of uric acid, ascorbic acid, bilirubin, glutathione, creatinine, formalin, hemoglobin, tetracycline, l-cysteine, l-dopa, dopamine, methyldopa, and citric acid can cause  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ Ø Advantages o More accurate than the glucose 3. 2-Hour Post Prandial Blood Glucose oxidase mtd § Measures how well the body § Not affected by ascorbic metabolizes glucose acid or uric acid § Based on the principle that glucose o May be performed on serum or level n blood drawn 2 hours after a plasma collected using heparin, meal returns to normal in normal EDTA, fluoride, oxalate, or citrate individuals while remains o Other samples may be used: significantly increased in diabetic urine, CSF, and serous fluids patients. Ø Disadvantages § Procedure o Gross hemolysis and icterisia may o Patient must eat a cause a FALSE DECREASE in complete meal (75 grams results. of glucose) OR a solution containing 75 g of glucose § NONSPECIFIC- it reacts is administered with any sugars with six o Specimen for plasma carbon units (fructose, glucose measurement is galactose, glucose) drawn 2 hours later o RBCs contain glucose-6- § Reference value must be phosphate and intracellular 200 mg/dL and is confirmed on a subsequent day by § hemolyzed samples either an increased random or require a serum blank fasting glucose level, the patient is correction (subtraction of diagnosed with diabetes the reaction rate with hexokinase omitted from 4. Glucose Tolerance Test (GTT) the reagent) § Multiple blood and urine glucose test § Also referred to as CHALLENGE TEST LABORATORY DETERMINATIONS OF GLUCOSE § Used to diagnose Gestational 1. Random Blood Glucose (RBG) Diabetes Mellitus Ø MONITORING TEST for blood glucose § used to determine how well the body metabolizes glucose over a required Ø For determination of glucose at anytime of the day period of time Ø Requested during: § Based on the principle that, a normal individual when given a glucose a. Insulin Shock challenge is capable of converting it b. Hyperglycemic Ketonic Coma to glycogen (for storage) and blood glucose returns to normal after 2 c. EMERGENCY CASES J hours. 2. Fasting Blood Glucose (FBG) § Diabetic patients will remove glucose Ø SCREENING TEST for serum glucose in from the circulation at a slower rate the diagnosis of Diabetes Mellitus due to insulin deficiency : Fastin Ø NPO (non-per-orem) for 8-10 hours 5. GLYCOSYLATED HEMOGLOBIN (GLYCATED HEMOGLOBIN) (HbA1C) § Now the PREFERRED TEST FOR THE ASSESSMENT OF GLYCEMIC CONTROL § for monitoring of LONG TERM GLUCOSE CONTROL (2–3 months)  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ § Sample of choice: o insulinoma = ↑ C-peptide and ↑ insulin o non fasting WHOLE BLOOD drawn in EDTA o injected or exogenous insulin = ↓ tube C-peptide and ↑ insulin § Reference range: 4-6%, 6.5% on more than two occasions is indicative of § Specimen: Diabetes Mellitus WHOLE BLOOD CAPILLARY GLUCOSE § For every 1% change in HbA1C § Uses a GLUCOMETER value, 35 mg/dL is added to plasma glucose level § Hematocrit affects POCT glucose measurements. § Disadvantage: Falsely low values observed in hemolytic anemias o High hematocrit = lower glucose (sickle cell, hemoglobinopathies, o RBC glucose concentration is etc.) lower than plasma 6. FRUCTOSAMINE (GLYCATED ALBUMIN/ concentration GLYCOSYLATED ALBUMIN) § NOT used to diagnose diabetes or § MOST WIDELY USED TO ASSESS hypoglycemic disorders SHORT-TERM GLYCEMIC § For confirmation, laboratory measures of CONTROL plasma glucose is required because of § Used in monitoring glucose level higher accuracy at a shorter time interval (3-6 weeks) § INBORN ERROR OF METABOLISM 1. Galactosemia INTERPRETATION OF RESULTS (WHO) § Congenital deficiency of one of 1. FOR FASTING BLOOD GLUCOSE the three enzymes involved in Non Diabetic: < 100mg/dL the conversion of galactose into glucose. Impaired FBS: > 100mg/dl but < 126 mg/dL 100 - 126 Diabetes Mellitus: > 126 mg/dL o Galactose-1-phosphate uridyl transferase 2. FOR ORAL GLUCOSE TOLERANCE TEST o Galactokinase Normal OGTT (2hr Glucose): < 140 mg/dL o Uridine diphosphate Impaired OGTT (2hr Glucose): 140-199 galactose 4-epimerase mg/dL Diabetes Mellitus (2hr Glucose): > 200 § Since galactose cannot be mg/dL converted into glucose, in accumulates in the blood and § DIAGNOSTIC CRITERIA FOR DIABETES urine MELLITUS 2. Essential fructosuria o RBG: > 200 mg/dL with symptoms of DM § a genetic disorder charaterized by accumulation of fructose in blood and o FBG: > 126 mg/dL urine due to lack of fructokinase o 2-hr Post Prandial: > 200 mg/dL 3. Hereditary fructose intolerance o HbA1c : > 6.5% § inborn error of fructose metabolism caused by a deficiency of the enzyme MISCELLANEOUS TEST FOR GLUCOSE aldolase B Anion Gap - used to monitor recovery 4. Fructose-1,6-biphosphate deficiency from DKA 5. Glycogen storage diseases (GSD) C-peptide - used to identify the cause of hypoglycemia  MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 1 CLinical chemistry 1 MR. nickson r. patawaran, RMT | CARBOHYDRATES | MTAP 1 MIDTERMS ____________________________________________________________________________________ § Due to inherited deficiencies of enzymes that control the synthesis or breakdown of glycogen § Glycogen storage diseases that primarily affect the liver usually manifest with hypoglycemia and hepatomegaly, whereas those affecting muscle commonly cause muscle cramps, weakness, fatigue, and exercise intolerance.