Clinical Chemistry Lab - 04 Carbohydrate Methodologies

o CLINICAL CHEMISTRY LABORATORY CARBOHYDRATE METHODOLOGIES GLUCOSE METHODOLOGIES CHEMICAL METHODS SPECIMEN CONSIDERATION • • • Standard Specimen: venous serum o Plasma (5% lower than serum) o Whole Blood (15% lower than serum/plasma) o Capillary ▪ FBS: 7mg/dL higher than venous collected sample ▪ Glucose challenge: 20-70 mg/dL (20-25%) higher than concurrently drawn venous blood o Arterial blood (same as capillary blood) o CSF (approx. 60% of plasma concentration) o Peritoneal fluid (same as plasma glucose conc.) Infants – have lower glucose level than adults (due to decrease glycogen reserve) Plasma glucose increase with age: o Fasting: 2 mg/dL/decade o Postprandial: 4mg/dL/decade ▪ Glucose after meal o Glucose challenge: 8-13 mg/dL/decade SPECIMEN HANDLING & STORAGE • • • • • • • • Serum should be separated from the cells within 30 mins. . o If delay is expected (>30 minutes): use sodium fluoride preservative At room temp (20-25 C ̊ ): 7 mg/dL/hour decrease (glycolysis) in normal uncentrifuged coagulated blood At refrigerated temp (4 ̊C): 2 mg/dL/hour decrease (glycolysis) With long-term specimen storage (-20 C ̊ ), glucose values decrease significantly and progressively WBC and RBC: metabolize glucose resulting to decrease value in clotted uncentrifuged blood Leukocytosis: excess glycolysis o Increased leukocytes Sterile non-hemolyzed serum is stable for 8 hours (25 C ̊ ) or 72 hours (4 C ̊ ) In serum specimen (without bacterial contamination or leukocytosis) o 90 minutes delay on separation of serum from cells is acceptable. o Not usually followed OXIDATION-REDUCTION METHOD ALKALINE COPPER REDUCTION METHOD • • • • • Used to inhibit glycolysis by inhibiting the action of enolase. 2 mg NaF/mL of blood - recommended concentration Enolase - an important enzyme in glycolysis that uses Mg2+ as cofactor. Fluoride forms ionic complex with Mg2+. Weak anticoagulant: binds Ca2+ (but clot may occur after few hours). Potassium oxalate is added to enhance anticoagulant activity o 2 mg K2C2O4/mL of blood - recommended Samples collected in these tubes should not be used for measurement of other analytes: o Fluoride ion inhibits urease activity – increased urea Principle: Reduction of cupric ions (Cu2+) to cuprous ions (Cu+) forming cuprous (Cu2O) in hot alkaline solution by glucose. o • • • • Cuprous ions are reacted with other substances to form chromogenic solutions/products Folin Wu Method Cuprous ions + phosphomolybdate ↓ phosphomolybdic acid or phosphomolybdenum blue Nelson Somogyi Method Cuprous ions + arsenomolybdate ↓ arsenomolybdic acid or arsenomolybdenum blue Neocuproine Method (2,9-dimethyl-1,10-phenantroline hydrochloride) Cuprous ions + neocuproine ↓ Cuprous − Neocuproine Complex (yellow or yellow orange) Benedict’s Method (Modification of Folin-Wu) o Used for the detection and quantitation of reducing substances in body fluids like blood and urine o Stabilizing agent: Citrate or tartrate o Copper (II) hydroxide + glucose → gluconic acid + Cuprous ion + water ▪ Cuprous ion - turn blue into red precipitate ALKALINE FERRIC REDUCTION METHOD • • • • SODIUM FLUORIDE • • • Potassium oxalate causes cell water loss thereby diluting the plasma. ▪ Water is extravagated ▪ Concentration of analytes are diluted aka Hagedorn Jensen Titrimetric method Inverse Colorimetry o Initial solution is chromogenic (colored), and color disappears when reacted with glucos, Principle: Potassium ferricyanide (yellow) is reduced to potassium ferrocyanide (colorless) when heated with alkaline solution by reducing sugars CONDENSATION METHOD DUBOWSKI METHOD • • • Use O-toluidine (aromatic amine) as substrate Principle: Glucose can form chromogenic Schiff’s bases w/ aromatic amines when heated with acidic solution (630 nm). Interferences: Galactose and Mannose ENZYMATIC METHODS • Most widely used DRY SLIDE ANALYZER • • GLUCOSE OXIDASE METHOD • • • • The most specific enzyme reacting with only β-D-Glucose. o 64% of glucose is β-D-Glucose o 34% is α-D-Glucose o Mutarotase converts α-D-Glucose to β-D-Glucose ▪ Mutarotation - conversion process Also measures CSF and Urine glucose Most glucose oxidase preparation contains catalase that decomposes peroxides thus reducing the final color of the end-product. Interferences: o False decrease: ascorbic acid, uric acid, bilirubin, hemoglobin, tetracycline, glutathione o False increase: bleach • • Glucose is measured using glucose oxidase method. 10 uL of sample is placed on a porous film on top of the layer containing reagents. Glucose diffuses through the film and reacts with the reagent to product colored end-product. The intensity of colored product is measured by reflectance spectrophotometry. SAIFER GERNSTENFIELD METHOD • • • • Colorimetric Glucose Oxidase Method Colorimetric and enzymatic method. o Enzymatic - uses glucose oxidase o Colorimetric - colored product measured w/ spectrophotometer Coupling reaction using peroxidase o Initial rxn - use glucose oxidase o Next rxn - use peroxidase Chromogens: o 3-methyl-2-benzothiazolinone hydrazone o N,N-dimethylaniline o O-dianisidine HEXOKINASE METHOD • • • • • • Reference method for glucose determination More accurate than glucose oxidase Based on coupled enzyme reaction assay that uses hexokinase and glucose-6-phosphate dehydrogenase (G6-PD) o G-6-PD is highly specific and not affected by ascorbic acid or uric acid. ▪ Ascorbic and uric acid can interfere Serum and plasma may be used o Plasma collection in NaF with oxalate, EDTA, heparin, citrate Other samples: CSF, Urine, and Serous Fluid Interferences: hemoglobin, drugs, bilirubin, lipemia Principle: • • • • • NADP - Nicotinamide adenine dinucleotide phosphate NAD - Nicotinamide adenine dinucleotide Leuconostoc monteroides - bacteria NADPH - Nicotinamide adenine dinucleotide phosphate NADH - Nicotinamide adenine dinucleotide + Hydrogen • • Absorbance is directly proportional to conc. of glucose POLAROGRAPHIC GLUCOSE OXIDASE • • • • Not a colorimetric method, it a polarogrphic Measures oxygen consumption rate which is directly proportional to the amount of glucose present. o Amount of oxygen reduced = amount of glucose o H2O2 has tendency to convert back to oxygen H2O2 is prevented from converting back to O2 by addition of molybdate, iodide, catalase and ethanol. The enzymatic conversion of glucose is quantitated by the consumption of oxygen on an oxygen-sensing electrode. o Electrode measures oxygen consumption rate GLUCOSE DEHYDROGENASE METHOD • • • • • • Glucose is reduced to produce a chromophore that is measured spectrophotometrically or an electrical current. Catalyzed the oxidation of glucose to gluconolactone accompanied by reduction of NAD+ to NADH. Amount of NADH is proportional to glucose concentration. Mutarotase is added to shorten time necessary to reach equilibrium It provides results in close agreement with hexokinase method. Does not show interferences with common anticoagulants and normal serum analytes. • • SAMPLES FOR GLUCOSE MEASUREMENT • GLUCOSE DEHYDROGENASE • Spectrophotometry o Colorimetric method requiring colored substance o Mutarotase - shorten time to reach equilibrium o MTT - tetrazolium salt • • • Electrical Current o Measures electric impulses/electrons produced o PQQ - pyrroquinoline quinolone Results of this test provide information about glucose pattern over hours to days Supplemental but not a replacement for POCT blood glucose monitoring • • • Random Blood Sugar o Blood collected anytime of day usually during emergency cases o Requested during insulin shock and hyperglycemic ketonic coma o RV: <160 mg/dL (≥160 mg/dL RBS – FBS, HbA1c, or 2-hr OGTT should be performed) o Not included in the diabetes panel ▪ ≥200 mg/dL is a sign of DM. Fasting Blood Sugar o It is a measure of overall glucose homeostasis o Requirement: NPO at least 8 hours before the test. ▪ NPO - no food or drink intake o RV: 70-99 mg/dL 2hour Postprandial Blood Sugar o It measures how well the body metabolize glucose. o 2 hours after meal o Uses standardizes glucose load (75grams glucose) o RV: 60-140 mg/dL Glucose Tolerance Test Glycosylated Hemoglobin (HbA1c) Fructosamine GLUCOSE TOLERANCE TEST • • • • DEXTROSTIX • • • • • • • aka Cellular strips (POCT) Important in establishing correct insulin amt for next dose Effective in reducing the rate of development of diabetic complications Employs enzymatic method (usually glucose oxidase method) Useful for detection of hyperglycemia and hypoglycemia among diabetic patient. Capillary blood glucose: ≥140 mg/dL o Should be rescreened with FBS, HbA1c, or OGTT using venous sample. Should not be used to diagnose diabetes. INTERSTITIAL GLUCOSE MEASURING DEVICE • • Used for continuous monitoring of glucose levels in people with diabetes. Utilized electrochemical method to automatically and frequently measure glucose levels in the interstitial fluids of dermis or subcutaneous fat tissues. • • Multiple blood sugar test It is used to determine how well the body metabolizes glucose over a required period of time. Should be performed to diagnose gestational diabetes. Not recommended for routine clinical use in the diagnosis of diabetes. ** Confirmatory: DM, GDM, Impaired FBG 2 Kinds: o Oral Glucose Tolerance Test (OGTT) o Intravenous Glucose Tolerance Test (IVGTT) ORAL GLUCOSE TOLERANCE TEST • • • Two Methods: Janney-Isaacson Method (Single dose method) – most common o Steps: 1. Extract initial blood glucose (FBS) 2. Instruct patient to drink glucose load (w/in 5 mins) 3. Collect blood samples; 1-hour, 2-hour, 3-hour post glucose load. (total of 4 blood samples) o Urine may also be collected Exton Rose Method (Divided Oral Dose or Double Dose Method or 1 hour 2 dose method**) - 3 blood samples o Steps: 1. Draw blood for fasting blood glucose 2. Give half of a 100grams glucose drink (1st dose) 3. Draw blood after 30 mins. of 1st dose glucose load 4. Give the remaining half of the 100 grams glucose drink (2nd dose) 5. Draw blood after 30 mins of 2nd dose glucose load • Requirements for OGTT 1. Patient should be ambulatory (mobile, active px) ▪ Only CC test requiring ambulatory pax ▪ Only CC test who ** 2. Unrestricted diet of 150g carbohydrates/day for 3 days prior to testing. 3. The patient should be restricted from smoking or alcohol intake prior to testing (affect glucose conc.) 4. Fasting for 8-12 hours (for baseline sample) 5. Glucose load: ▪ 75 grams – WHO standard glucose load ▪ 100 grams – pregnant; as requested ▪ 1.75 g of glucose/KBW – children (max. of 75 g glucose) • INTRAVENOUS GTT • • • • • • Used for DM with gastrointestinal disorders. For patients with malabsorption disorders o Glucose drink will not be absorbed Glucose load: 0.5 g glucose/Kbw (administered intravenously within 3 minutes) FBS should be collected before administration 2nd Blood Collection: 5 minutes after IV glucose load. o to measure success of IV administration Collect blood: 1-hr, 2-hr, 3-hr post IV glucose loading. Relationship between HbA1c and Plasma Glucose o Estimated average glucose (eAG) can be calculated from HbA1C. (eAG = 2-4 mos.) o eAG = 28.7 x HbA1c – 46.7 o For every 1% change in HbA1C = approx. 35 mg/dL change in plasma glucose HbA1c 5 6 7 8 9 10 11 12 REFERENCE VALUES (OGTT) MEASUREMENT • • • • Confirmatory Sample: 2nd hour post-glucose load sample 2 hours after glucose load, blood glucose should return to the fasting level. o 1st hour sample - control sample o Samples should be decreasing every hour 2-hr OGTT RV: <140 mg/Dl • • • GLYCOSYLATED HEMOGLOBIN (HbA1c) • • • • • • • aka glycated hemoglobin The largest fraction of normal hemoglobin A (both diabetic and non-diabetic) Weighted average of glucose level (2-4 months) o young RBC as the main contributor Dependent on RBC life span and average plasma glucose Preferred test to assess long-term glycemic control DM patients: HbA1C for every 3-6 months (using NGSPcertified method) o DM patient with stable glycemic control: HbA1c at least twice a year. o DM patient w/ unstable glycemic control: HbA1c quarterly Synthesis of HbA1c 1. Glucose attach to one or both N-terminal valines of βchain of hemoglobin forming labile almidine 2. Labile almidine undergo Amadori rearrangement which then converted into stable ketoamine (HbA1c). ▪ RBC lifespan, Hgb is in RBC, shorter life span, Hgb are extravagated decreasing HbA1c level ▪ Plasma glucose concentration attaches to Hgb eAG (mg/dL) 97 126 154 183 212 240 269 298 Specimen: EDTA whole blood (w/ or w/o fasting) o Only CC test that relies on RBC lifespan/lysis 1st step: Direct lysis of RBC to form hemosylate (solution of hemolyzed blood) 1st reagent: lysing agent Methods: o Affinity chromatography – preferred method of choice ▪ Not affected by Hgb F, S and C. o Cation-exchange chromatography ▪ Temperature dependent ▪ False increased: + Hgb F ▪ False decreased: + Hgb S and C o HPLC - all forms of hgb can be separated o Electrophoresis o Latex immunoagglutination inhibition methodology ▪ Concentration of HbA1c & total Hgb is measured ▪ Expressed as percent HbA1c per total Hgb. DIAGNOSTIC VALUES (HbA1c) • • • • • Reference Value: 3.0 - 6.0% Pre-diabetes: 5.7 – 6.4% Diabetes: ≥6.5% (on two occasions using NGSP-certified method) Increased HbA1c: o Older RBC (higher chance of glycosylated HbA) o IDA o Hemoglobin F Decreased HbA1c: o Shortened RBC lifespan (e.g. hemoglobinopathies) o Vitamin C and E. o Hemoglobin C and S. o Hemolysis ▪ Patient with hemolytic anemia – not allowed for HbA1c test. FRUCTOSAMINE • • • • • • • • KETONE TEST aka glycosylated or glycated albumin, plasma protein ketoamine Most widely used to assess short-term glycemic control (36 weeks) o Depends on albumin lifespan Specimen: Serum (w/ or w/o fasting) Should not be used in cases of hypoalbuminemia (≤30 g/L) Methods: HPLC, Affinity chromatography, photometric assay Useful for patients with decreased RBC life span: o Chronic hemolytic anemia o Hemoglobin C and S. Interferences: high uric acid, triglyceride and bilirubin levels; heparin; hemolysis RV: 205-285 μmol/L DIAGNOSTIC CRITERIA Normal/ Prediabetes Impaired Plasma Glucose Diabetes Mellitus Impaired Glucose Tolerance Gestational Diabetes Mellitus Hypoglycemia • • • • • • • • • • • • • • • • • FBS 70/99 mg/dL 2hr-GTT <140 mg/dL 100-125 mg/dL - ≥126 mg/dL ≥200 mg/dL - 140-199 mg/dL ≥92 mg/dL ≥153 mg/dL ≤60 mg/dL - HbA1c <3-4% (prediabetes: 5-7%) - RBS <160 mg/dL (prediabetes: ≥160 mg/dL) - ≥6.5% *using NGCP-certified assay - ≥200 mg/dL (with symptoms of DM) - 1 hr Glucose Challenge Test: ≥180 mg/dL - Prediabetic numbers should be reassessed w/ FBS, GTT Impaired plasma glucose - conc. of FBS bet. normal and diabetic Glucose challenge test 1. Patient should be on non-fasted state (routine sample only) 2. Prior to collection, instruct patient to have glucose load intake of 50g. 3. After 1 hour of glucose load intake, collect blood. C-PEPTIDE TEST Formed during the conversion of pro-insulin to insulin The amount of circulating C-peptide provides reliable indicators for pancreatic and insulin secretions (β-cell function) Used to monitor individual responses to pancreatic surgery The test mainly evaluates hypoglycemia and continuous assessment of β-cell function Specimen: fasting serum Methodology: immunometric assay Increased: insulinoma, type 2 DM, hypoglycemic drugs Decreased: Type 1 DM Reference value: 0.90-4.3 ng/mL (CF to nmol/L: 0.333) Normal C-peptide:insulin ratio: 5-15:1 Ketone bodies are produced by liver through metabolism of fatty acids to provide energy source at times of low carbohydrate source Recommended when plasma glucose reached 300 mg/dL 3 ketone bodies: o β-hydroxybutyric acid (78%) o Acetoacetic acid (20%) o Acetone (2%) – increased in CHO metabolism defects o β-hydroxybutyric acid & Acetoacetic acid ▪ normal ratio = 1:1; 0.5-1.0 mmol/L each ▪ increased β-hydroxybutyric acid to acetoacetic acid ratio seen in diabetic ketoacidosis (CKA) β-hydroxybutyric acid increased in DKA & fall w/ treatment. Acetoacetic acid and acetone levels rise with treatment. Calculation of anion gap is used to monitor recovery from ketoacidosis. Specimen: fresh urine or serum (important for individuals with type 1 DM to detect ketosis) KETONE METHODOLOGIES • • • - OTHER TESTS • • • • • Gerhardt’s ferric chloride test – reacts only with acetoacetate o many interferences (e.g. salicylates) Nitroprusside test – 10x more sensitive to acetoacetate than acetone. o Addition of glycerin allows acetone detection Acetest tablets – detects acetoacetate and acetone (lesser degree) o Uses nitroprusside method o Tablet composition: Glycerin, sodium nitroprusside, disodium phosphate, lactose. o Suitable for urine ketone determination Ketostix – modification of Acetest o Use reagent strips rather than tablets o Suitable for urine ketone determination KetoSite Assay – detects β-hydroxybutyric acid MICROALBUMINURIA • • • • • • • • • Urinary albumin excretion of 30-300 ug/mg creatinine Not detected by urine reagent strips. Useful to assist in diagnosis of diabetic kidney disease. Early indicator of glomerular dysfunction associated with Type 1 DM. (+) microalbuminuria: 2 of 3 specimens collected within 6 months are abnormal DM patient: annual urinary albumin excretion is recommended Method: Random-spot albumin-creatinine ratio Reference value: 0-29 ug/mg creatinine Clinical albuminuria: >300 ug/mg creatinine o Detected by urine reagent strips INTRAVENOUS GALACTOSE TOLERANCE TEST • • For type 1 GSD (hypoglycemia) Serial blood specimen are collected for 2 hours at 15 minutes interval for testing.