MLS Review - CC - Carbohydrates & Glucose

MEDICAL LABORATORY SCIENCE REVIEW • CLINICAL CHEMISTRY: CARBOHYDRATES, GLUCOSE, AND RELATED METHODOLOGIES AND CONDITIONS OUTLINE • Carbohydrates • o Classification of Carbohydrates o Glucose o Pancreas • Clinical Conditions Associated with Carbohydrate Metabolism o Hypoglycemia o Hyperglycemia • o Diabetes Mellitus Glucose Methodologies o Specimen Consideration o Specimen Handling & Storage o Chemical Methods o Enzymatic Method o Glycosylated Hemoglobin Methods o Self-Monitoring of Glucose Inborn Errors Of Carbohydrate Metabolism DISACCHARIDES • • • CARBOHYDRATES • • • • Aka as starch/sugar Aldehydes/ketone derivatives of polyhydroxy alcohols Consists of C, H, O Major food and energy source of the body CLASSIFICATION OF CARBOHYDRATES • Examples of Glucose: o Glucose - most abundant ▪ Measured for blood sugar o Fructose - found in seminal fluid o Galactose o Ribose/deoxyribose - found in nucleic acids ▪ RNA (ribose) & DNA (deoxyribose) 2 monosaccharide units joined by glycosidic bonds/linkages 2 types of glycosidic linkages o Alpha (α) - oriented down o Beta (β) - oriented up Examples: o Sucrose - glucose + fructose ▪ aka table sugar ▪ Has α glycosidic linkage o Lactose - glucose + galactose ▪ Principal disaccharide in milk (in cows, humans) ▪ Has β glycosidic linkage o Maltose - glucose + glucose ▪ Has α glycosidic linkage Carbohydrates can be classified based on the number of saccharide units MONOSACCHARIDES • • Simplest carbohydrates Characterized by: o Functional Group ▪ Aldose = Aldehyde • R=OH • Functional group is at the end ▪ Ketose = Ketone • Functional group is between two carbons • o POLYSACCHARIDES • • Has 3 or more monosaccharide units Examples: o Cellulose - found nearly in plant cells ▪ Indigestible by humans ▪ Repeating glucose unit ▪ Has β glycosidic linkages ▪ Has straight chain structure = unbranched polysaccharide o Glycogen ▪ Major form found in animals and humans ▪ Majority are stored in the liver • Some are found in the muscle ▪ Has repeating glucose units ▪ Branched polysaccharide o Starch ▪ Main polysaccharide found in seeds/roots of plants ▪ Amylose (20%) - unbranched ▪ Amylopectin (80%) - branched Aldohexose Ketohexose Aldo = fxnal group at end Keto = between 2C Hexose = 6 carbon Hexose = 6C Number of Carbon Atoms ▪ Monosaccharide may contain 3-6 carbon atoms only • 3 - triose • 4 - tetrose • 5 - pentose • 6 - hexose 1 GLUCOSE • • • • • • Primary energy source of human The only carbohydrate directly used as energy o Other carbohydrates must be biochemically processed by the body to be used as energy source Stored as glycogen (repeating units of glucose) o Stored in liver (majority) and muscles For glucose to be used, it must be within the cell (glycolysis) Does not freely enter the cell o Insulin - allow glucose to enter the cell Most medically important carbohydrate o Brain function is majorly dependent on glucose ▪ ↓ BG = impaired brain function ▪ 1/2 to 2/3 of glucose → utilized by brain during resting/sleeping phases • • PANCREAS • • • METABOLISM OF GLUCOSE • Pathways of Glucose Metabolism Ketones - byproducts from using fats as energy source (ketosis) Continuous increased ketones can lead to coma • Effects on Glucose Level Glycolysis Metabolism of glucose to Decreased form energy blood glucose Gluconeogenesis Formation of glucose from Increase blood non-carbohydrate source glucose (protein, amino acids, lipids) Glycogenolysis Breakdown of glycogen to Increase blood produce glucose glucose Glycogenesis Formation of glycogen to Decreased store excess glucose; blood glucose conversion of multiple glucose units to glycogen Lipogenesis Conversion of Decreased carbohydrate to fatty acids blood glucose (CHO → FA) Lipolysis Decomposition of fats Increased (TAG) when glucose is blood glucose absent; fats are used as alternative source of energy; (TAG → 3FA + Action Glycerol → gluconeogenesis) • Major organ that regulates glucose in the blood Can maintain glucose based on substances it produces Exocrine: Produce and secretes amylase o Amylase - breaks down polysaccharides by destroying glycosidic bonds ▪ Smallest enzyme that can pass through glomerulus • Normally present in the urine ▪ Earliest pancreatic marker for pancreatic dse ▪ Performed STAT for ER admissions • To rule our pancreatic dse/issue ▪ S-type (ptyalin) - produced by salivary glands ▪ P-type (amylopsin) - produced by the acinar cells in the pancreas Endocrine (produced in islet of Langerhans in pancreas) o Insulin - produced by β cells of islet of Langerhans ▪ Primary hormone responsible for the entry of glucose into the cell; (liver, muscle, & adipose tx) • Initiates entry of glucose into the cell ▪ Enhances the permeability of cell to glucose ▪ Only hypoglycemic agent/hormone ▪ Promotes: glycogenesis, lipogenesis, & glycolysis • Decreases blood glucose ▪ Decreases: glycogenolysis ▪ Sample must be free from hemolysis • Hemolysis = false ↓ insulin (due to presence of insulin degrading enzymes found in RBCs) ▪ Injury/issues in the pancreas = ↓ insulin production = ↑ glucose levels o Glucagon - produced by α cells of islet of Langerhans ▪ Primary hyperglycemic agent ▪ Prevents cellular uptake of glucose • Prevents entry of glucose into the cell ▪ Released during stress and fasting states • Stress may lead to hyperglycemia ▪ Promotes: glycogenolysis, gluconeogenesis ▪ Reference value: 20-50 pg/mL o Somatostatin - delta (δ) cells of islet of Langerhans Other Hyperglycemic Hormones o Glucocorticoids ▪ Produced in zona fasciculata of adrenal cortex ▪ Eg. cortisol ▪ Promotes: gluconeogenesis, lipolysis ▪ Decreases cellular entry of glucose o Catecholamines ▪ Secreted by chromaffin cells in adrenal medulla ▪ Eg. dopamine, epinephrine, ▪ Inhibits insulin secretions ▪ Promotes: glycogenolysis, lipolysis ▪ Epinephrine - released during stress o Growth hormones/ Somatotropin ▪ Secreted by anterior pituitary gland/adenohypophysis ▪ Promotes: glycogenolysis, glycolysis ▪ Decreases cellular entry of glucose o Thyroid Hormones (T3, T4) ▪ T3 - triiodothyronine ▪ T4 - thyroxine, tetraiodothyronine ▪ Promotes: glycogenolysis, gluconeogenesis,. Intestinal absorption of glucose ▪ Increases blood glucose 2 o o Adrenocorticotropic hormone (ACTH) ▪ A glucocorticoid (increases blood glucose) ▪ Secreted by anterior pituitary gland ▪ Stimulates release of cortisol by adrenal cortex • Cortisol = glucocorticoids • ↑ ACTH = ↑ glucocorticoids = ↑ glucose ▪ Promotes: glycogenolysic and gluconeogenesis Somastostatin ▪ secrete by delta cells of Islets of Langerhans ▪ Synthesized by paraventricular & arcuate nuclei of the hypothalamus ▪ Inhibits action of insulin and/or glucagon • Glucose may increase if glucagon is inhibited as there are other glycemic agents • • HORMONAL REGULATION OF GLUCOSE Hormones Modes of action Insulin Initiates cellular uptake of glucose; only hypoglycemic agent Glucagon Prevents cellular uptake of glucose Glucocorticoids Decreases cellular entry of glucose Catecholamines Inhibits insulin secretions Growth hormones Decreases cellular entry of glucose Thyroid Hormone Promotes intestinal absorption of glucose Adrenocorticotrophic Stimulates release of Hormone (ACTH) cortisol (glucocorticoid) Somatostatin Inhibits action of insulin and/or glucagon Effects on Glucose Decrease glucose Increase glucose Increase glucose Increase glucose Increase glucose Increase glucose Increase glucose Increase glucose CLINICAL CONDITIONS ASSOCIATED WITH CARBOHYDRATE METABOLISM HYPOGLYCEMIA • • • Decreased serum glucose concentration A life threatening condition o Brain is highly glucose-dependent o ↓ glucose cause impairment of brain functions o Symptoms mostly affect CNS Diagnostic Criteria: Whipple’s Triad • Symptoms: o Neurogenic – tremors, palpitations, anxiety, diaphoresis ▪ Andrenergic - affects autonomous nervous system ▪ Predominantly found in patients w/ reactive hypoglycemia ▪ Reactive Hypoglycemia - occur 4 hours after meal o Neuroglycopenic – dizziness, tingling, blurred vision, confusion, behavioral changes ▪ Impaired brain function due to decreased glucose in the brain (<40 mg/dL) ▪ Found in patients with fasting hypoglycemia ▪ Fasting Hypoglycemia - occur after 10 hours without food intake Hypoglycemic Values: 70-99 mg/dL Normal Fasting Blood Glucose (FBG) 65-70 mg/dL Glucagon and other hyperglycemic hormones are released into the circulation (to promote increase of plasma glucose) ≤60 mg/dL Strongly suggests hypoglycemia (when found in series of glucose testing); requires Whipple’s triad to confirm 55 mg/dL Observable symptoms of hypoglycemia appear 50 mg/dL Impairment of cerebral function begins Causes of Hypoglycemia o Alcohol consumption ▪ Alcohol can inhibit hepatic gluconeogenesis (conversion of other macromolecules to glucose) ▪ Alcohol can increase glycogen phosphorylase activity (Involved in glycogenesis) • Increases glycogenesis (glucose → glycogen) o Severe sepsis o End-stage Renal Disease (ESRD) o Cortisol and/or Growth Hormone deficiencies o Alimentary Hypoglycemia (reactive hypoglycemia) ▪ Occur 4 hours after meal HYPERGLYCEMIA • • • • • Increased serum glucose concentration Value: >99 mg/dL glucose Toxic to beta-cell function o Beta cell function - insulin production Normally, insulin is produced after meal/glucose intake o Chronic hyperglycemia (prolonged cases) can result to impaired insulin production → ↓ insulin → failure to decrease blood glucose levels ▪ Insulin is the only hypoglycemic hormone General Laboratory Findings: o Increased urine and serum glucose o Increased urine specific gravity o Ketones in urine and serum ▪ Occur mostly in DM type I patients o Decreased blood and urine pH (acidosis) o Electrolyte imbalance (↓Na+, ↑K+, ↓HCO3) DIABETES MELLITUS • • 1. 2. 3. Low plasma glucose = ≤60 mg/dL Symptoms associated w/ hypoglycemia (mainly CNS) Glucose is raised and symptoms are relieved • Most common carbohydrate metabolism disorder A group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin secretion, insulin receptors, or both. FBS of ≥ 126 mg/dL on more than one testing 3 • • • Glucosuria - glucose in urine o Renal threshold for glucose = 180 mg/dL ▪ Once glucose levels reach the threshold, excess glucose will not be reabsorbed by the kidneys and is excreted with the urine Ketosis o Occur if the body metabolizes or uses noncarbohydrate source to form energy (eg. lipids) o Conversion of lipids to form energy, ketones are produced ▪ Defects in insulin secretion/production negatively affect cellular uptake of glucose by inhibiting use of glucose as energy source → lipids are used for energy production → ketone byproduct o Ketones - byproduct of ketosis ▪ β-hydroxybutyrate to acetoacetate ratio (ketone ratio) • Normal = 1:1 • Severe DM = 6:1 ▪ Reversible through insulin administration • Allows utilization of glucose by cells General Signs & Symptoms: o Polyuria, polydipsia, polyphagia (3Ps) ▪ Polyuria - increase in urination, due to polydipsia ▪ Polydipsia - excessive water intake • ↑ Blood glucose = ↑ osmolality = ↑ thirst o Thirst = compensation to ↑ osmolality ▪ Polyphagia - increased hunger • Due to false sensation of need to eat for energy as glucose is not efficiently utilized o Rapid weight loss o Hyperventilation o Mental confusion and possible loss of consciousness TYPE 1 VS TYPE 2 DIABETES MELLITUS TYPE 1 DM Other Names Insulin Dependent Diabetes Mellitus (IDDM); Juvenile Onset DM; Brittle Diabetes; Ketosis-Prone Diabetes TYPE 2 DM Non-Insulin Dependent Diabetes Mellitus (NIDDM); Adult-type/Mature onset DM; Stable diabetes; Ketosis-Resistant Diabetes; Receptor-Deficient DM Pathogenesis Beta-cell destruction Insulin resistance due (lead to insulinopenia/ to alteration/absence absolute insulin of insulin receptors deficiency); manifests if 80-90% of β-cells are destroyed Onset Any age group; Any age group; Most common to Most common among childhood/teen adults Risk Factors Autoimmunity Genetics, Race, (Type 1 DM is an Ethnicity, Obesity, autoimmune disease) Sedentary Lifestyle, Dyslipidemia, Hypertension, Polycystic Ovarian Syndrome (PCOS) Pre-diabetes (+) Autoantibodies (-) Autoantibodies Stage (normal C-Peptide Ketosis Medication • C-peptide o Formed when insulin is produced by β-cells o Also released with insulin o Specimen: fasting serum ▪ Routine sample has false ↑ • After meal, insulin + c-peptide is produced • Pro-insulin o Insulin + C-peptide o Produced in β-cells o Inactive when produced o Requires cleavage of insulin and c-peptide → insulin can be active and used • Type 1 DM o (+) autoantibodies ▪ Glutamic acid decarboxylase - common in adults ▪ Insulin autoabs - common in children ▪ Islet cells autoabs ▪ Tyrosine phosphatase 1A-2 & 1A-2B ▪ Insulonoma-assoc. protein 2 autoab ▪ Zinc transporter 8 autoab o Poorly controlled ketosis as ketosis is primary energy source ▪ Absence of insulin inhibits cellular uptake of glucose causing use of non-carbohydrate sources as primary sources of energy through ketosis • Type 2 DM o Has strong genetic predisposition o Geneticist’s nightmare because it doesn’t follow the classical hereditary patterns/transmission ▪ There is no absolute pattern of inheritance of DM o Symptoms can be milder than type 1 o Some patients may be asymptomatic o If uncontrolled, it can be converted to type 1 DM ▪ Hyperglycemia is toxic to cells → destruction of beta cells → inability to produce/release insulin ▪ Type 1 DM = destruction of beta-cells o Metformin - increases sensitivity of cells to insulin → insulin can be efficiently use to allow use of glucose IDIOPATHIC TYPE 1 DM • • Develop abruptly Develop gradually Cause is unknown It does not have β-cell autoantibodies and have episodic requirements for insulin replacement GESTATIONAL DIABETES MELLITUS (GDM) • • • blood sugar but at risk) Symptoms TYPE 1 DM TYPE 2 DM Decreased or Detectable Undetectable (no insulin = no C-peptide) Common, poorly Rare controlled Insulin Oral Agent (Metformin) • Only occurs among pregnant women o Woman diabetic prior to pregnancy is not classified as GDM A type of glucose intolerance with onset or first recognition during pregnancy o Can disappear after pregnancy o 30-40% of GDM converts to type 2 DM within 10 yrs o Women with GDM should be evaluated 6-12 wks after birth for possible development Disorder characterized by impaired ability to metabolize carbohydrate usually caused by a deficiency of insulin, metabolic or hormonal changes. Screening should be performed bet. 24-28 weeks of gestation 4 CRITERIA FOR DIAGNOSIS OF DM • FBS 2hr-GTT HbA1c RBS Normal/ 70-99 60-140 3-6% <160 mg/dL Prediabetes mg/dL mg/dL (<5.7%) Diabetes ≥126 mg/dL ≥200 mg/dL ≥6.5% ≥200 mg/dL (w/ Mellitus DM symptoms) Impaired Fasting 100-125 Glucose mg/dL Impaired 140-199 Glucose mg/dL Tolerance Hypoglycemia ≤60 mg/dL (confirm w/ Whipple’s Triad) • • • • Random Plasma Glucose o Aka Random Blood Sugar (RBS) o Requested during insulin shock and hyperglycemic ketonic coma o Reference Value: <160 mg/dL ▪ ≥160 mg/dL RBS → perform FBS/HbA1c/2hr OGTT for confirmation Fasting Plasma Glucose o Requires 8-12 hour fasting no longer than 16 hrs o Measure of overall glucose homeostasis o Affected by diurnal variations ▪ Glucose is generally higher in morning than afternoon ▪ Testing should be performed in the morning ▪ Afternoon = false decreased o Age is a factor for increasing levels ▪ Increase of 2 mg/dL per decade o Reference Value: 70-99 mg/dL o Diabetic Value: ≥126 mg/dL ▪ 100-125 mg/dL = impaired fasting glucose (IFG) 2 hour Post-Prandial Blood Glucose o 2 hour after start of the meal o Variation of the test: Uses standardized Glucose Load (75g of glucose load = equivalent to a complete meal) o Within the two hour period, the patient is prohibited from any glucose intake o Increases with age by 4 mg/dL per decade o Reference Value: 60-140 mg/dL ▪ >140 mg/dL = hyperglycemia o Diabetic Value: ≥200 mg/dL ▪ With ↑ FBS/RBS the next day = diabetic Glycosylated Hemoglobin (HbA1c/Hgb A1c) o Aka glycated hemoglobin ▪ Glucose is non-enzymatically attached to 1 or both β-chain of hemoglobin ▪ Glucose is attached to one or both terminal valine chain of both β-chains • Irreversible when there is a glucose attached o Preferred test to assess long-term glycemic control o Reflects average glucose level over the past 2-3 months o Dependent of the following: ▪ RBC life span • ↓ life span = ↓ HbA1c • Pxs with hemolytic anemia and hemoglobinopathies (↓ RBC life span) cannot be subjected to HbA1c tests (false ↓) ▪ Plasma glucose level o Reference Value: ▪ Adult: 4.0 - 6.0% • American Diabetes Association (ADA) = <5.7% ▪ Pediatrics: <7.5% o Diabetic Value: ≥6.5% o Pre-diabetes: 5.7-6.4% • Fructosamine o Aka glycosylated/glycated albumin, plasma protein ketoamine o Most widely used to assess short-term glycemic control (3-6 weeks) o Specimen: serum (measurement of albumin) ▪ With or without fasting o Useful for patients with decreased RBC life span: ▪ Chronic hemolytic anemia ▪ Hemoglobin C and S ▪ Hemolytic anemias and Hemoglobinopathies o Not performed for px with hypoalbunemia (↓ albumin) ▪ ≤30 g/dL albumin = false ↓ o Methods: HPLC, Affinity chromatography, photometric assay o Interferences: high uric acid, triglyceride and bilirubin levels; heparin; hemolysis; o Reference Value: 205-285 umol/L Glucose Tolerance Test o Aka multiple sugar test o Requires multiple samples (2-4 blood samples) o It is used to determine how well the body metabolizes glucose over a required period of time. o Recommended for diagnosis of GDM o Not for routine diagnosis for DM ▪ Due to inconvenience of the test for patients (multiple samples taken) o Oral Glucose Tolerance Test ▪ Recommended Method for Non-Pregnant Patients: 1. No food intake for at least 10 hours 2. Blood sample #1 is taken after fasting (FBS) 3. Standardized Glucose Load is administered (75g of glucose load) 4. Blood sample #2 is taken 2 hours later o Reference sample for testing ▪ Total of 2 blood samples ▪ Reference Value: 2 hr (#2), 60-140 mg/dL • 140-199 mg/dL = impaired glucose tolerance ▪ Diabetic Value: 2 hr (#2), ≥200 mg/dL ▪ Requirements for Pregnant/Non-Pregnant: • Patient should be ambulatory (able to walk) o Only test that requires ambulatory pxs o Only test not performed for bed-ridden px • Unrestricted diet of 150g carbohydrates/day for 3 days prior to testing o Normal to high carbohydrate diet • Restrictions: exercise, food intake, drinking, smoking/alcohol (↑ BG) o Only water is allowed • Fasting for at least 10 hours • Must be performed in the morning o Affected by hormonal dilution due to circadian rhythm • Glucose load: o 75g – WHO standard glucose load o 100g – pregnant; as requested o 1.75g of glucose/KBW – children ▪ Max of 75 grams 5 o o Intravenous Glucose Tolerance Test (IVGTT) ▪ Performed on patients with gastrointestinal disorders (malabsorption syndrome) ▪ Glucose Load: 0.5g/KBW • Administered intravenously within 3 minutes ▪ Blood Collection (5 collection but 4 is used): 1. Fasting Blood Glucose (Spx #1) 2. IV administration of glucose load w/in 3 mins 3. 5 minutes after glucose administration (Spx #2) o Determine/confirm if IV glucose is successfully administered o Has no diagnostic significance, not used for testing (discarded for testing) 4. 1 hour, 2 hours, and 3 hours after glucose load (Spx #3, #4, #5) CRITERIA FOR DIAGNOSIS OF GESTATIONAL DM • • Criteria for Diagnosis of Gestational Diabetes Mellitus o IADPSG Consensus (One-step) (1/3 criteria is met) ▪ FBS: ≥92 mg/dL ▪ 1 Hr-GTT: ≥180 mg/dL ▪ 2 Hr-GTT: ≥153 mg/dL o NIH Consensus (Two-step) ▪ Screening (50g): • 1-hr: ≥140 mg/dL ▪ 100g OGTT (2/4 criteria is met) • FBS: ≥95 mg/dL • 1 Hr-GTT: ≥180 mg/dL • 2 Hr-GTT: ≥155 mg/dL • 3 Hr-GTT: ≥140 mg/dL Oral Glucose Tolerance Test o Recommended method o Methods for Pregnant Women: ▪ Old Methods • Janney-Isaacson Method • Exton Rose Method ▪ New Methods: • One-Step Method (IADPSG) • Two-Step Method (NIH) o Janney-Isaacson Method ▪ Single-dose method ▪ Uses 75g glucose lose ▪ Blood collection (4 samples) 1. Fasting for at least 10 hours 2. Blood sample #1 is taken after fasting (FBS) 3. Administer Standardized Glucose Load (75g of glucose load) o Px can’t eat after glucose load 4. Collect 1-hour (#2), 2-hour (#3), 3-hr (#4) samples after glucose load o Exton Rose Method ▪ Double-dose method ▪ Divided oral dose ▪ Uses 100g glucose load • Divided into two ▪ Blood collection (3 samples) 1. Fasting for at least 10 hours 2. Blood sample #1 is taken after fasting (FBS) 3. Administer half of glucose load (50g) 4. Collect blood spx #2 after 30 mins 5. Administer remaining half of glucose load (50g) 6. Collect blood spx #3 after 30 mins o One-Step Method (IADPSG) ▪ Recommended by International Association of Diabetes and Pregnant Study Groups (IADPSG) ▪ Performed among pregnant women at 24-28 weeks age of gestation who are not previously diagnosed with DM ▪ Steps: (3 samples) 1. Patient is fasting for 8 hours 2. Extract initial blood glucose (FBS) (spx #1) 3. Instruct patient to drink glucose load (75g) 4. Collect blood samples after: o 1-hour (spx #2) o 2-hours (spx #3) ▪ GDM can be diagnosed if one (1) of the following is met: • FBS: ≥92 mg/dL • 1-hr: ≥180 mg/dL • 2-hr: ≥153 mg/dL Two-Step Method (NIH) ▪ Recommended by National Institute of Health (NIH) ▪ Requires screening • Performed routinely (no fasting required) ▪ Steps: • Screening: Performed glucose measurement of blood sample collected 1 hour after 50g glucose load o Positive = ≥140 mg/dL o (+) screening proceed to 100g OGTT • 100g OGTT: performed next/another day after screening (4 samples) o Patient is fasting for 8 hours o Extract initial blood glucose (FBS) (spx #1) o Instruct patient to drink glucose load (100g) o Collect blood samples after: ▪ 1-hour (spx #2) ▪ 2-hours (spx #3) ▪ 3 hours (spx #4) • GDM can be diagnosed if two (2) of the following is met: o FBS: ≥95 mg/dL o 1-hr: ≥180 mg/dL o 2-hr: ≥155 mg/dL o 3-hr: ≥140 mg/dL OTHER TYPES OF DIABETES • • • • • Pancreatic disorders/Pancreatectomy o Removal of pancreas → no insulin production → impaired glucose cellular uptake → ↑ glucose Endocrine disorders o Cushing's syndrome: ↑ cortisol o Pheochromocytoma: ↑ catecholamines o Acromegaly: ↑ growth hormones o Hyperthyroidism: ↑ thyroid hormones o Increased hyperglycemic hormones increases glucose Drugs or chemical inducers of Beta cell dysfunction o Dilantin, pentamidine Drugs or chemical causing impairment of insulin action o Thiazides, glucocorticoids Genetic disorders o Down syndrome (trisomy 21) - immune defects may lead to type 1 DM o Klinefelter’s (XXY) - immune defects may lead to diabetes 6 o o o o • GLUCOSE METHODOLOGIES SPECIMEN CONSIDERATION • • • • • • • • Serum: standard specimen Plasma: 5% lower than serum Whole Blood: 10-15% lower than serum Capillary Blood o FBS: 7 mg/dL higher than serum o OGTT: 20-70 mg/dL (20-25%) higher than blood from venous blood Arterial Blood: differences similar with capillary blood CSF: Normal CSF glucose: 60-70% of plasma glucose Peritoneal Fluid: differences similar with serum & plasma Specimen Consideration Notes: o Plasma and serum are used interchangeably o Serum, plasma, & whole blood must not be collected on the arm of patients with IV fluids ▪ Some IV fluid may contain dextrose; 10% contamination of blood with 5% of dextrose can increase glucose by 500 mg/dL (false ↑) Folin Wu: Cu2+ + phosphomolybdic acid → phosphomolybdenum (blue) o Nelson Somogyi: Cu2+ + arsenomolybdic acid → phosphomolybdenum (blue) o Neocuproine (2,9-dimethyl-1,10-phenantroline HCl): Cu2+ + neocuproine → Cu − neocuproine complex (yellow/yellow-orange) o Benedict’s: Cu(OH)2 → Cuprous ion (brick red ppt) ▪ Benedict’s reagent - Cu(OH)2blue solution Alkaline Ferric Reduction Method o Aka Hagedorn Jensen o Titrimetric Method o Inverse Colorimetry - reduction of color is measured o Principle: o Rabson-Mendengall - severe insulin resistance (T2 DM) Leprechaunism - insulin resistance (type 2 DM) Huntington’s chorea - death of brain cells Turner syndrome - occur in females; partial/complete deletion of 1 X chromosome Exocrine disorders o Cystic fibrosis, neoplasia, hemochromatosis • • ▪ Potassium ferricyanide - yellow ▪ Potassium ferrocyanide - colorless ▪ Heated with alkaline solution Condensation Method o Uses O-toluidine (aromatic amine) o Principle: Glucose forms Schiff’s base w/ aromatic amine when heated w/ acidic solution (glacial HAc) ▪ Absorbance: 630 nm Interferences: galactose, mannose ▪ These sugars can form Schiff’s base as well ▪ o ENZYMATIC METHOD SPECIMEN HANDLING & STORAGE • • Serum/Plasma: o Separated from cells within 1 hour ▪ Cells metabolize glucose ▪ Delay may cause false decrease ▪ Leukocytosis (↑ WBC) exhibit excessive glycolysis → ↓ glucose o If delayed: use sodium fluoride additive (gray top) ▪ Sodium fluoride - weak anticoagulant that inhibit glycolysis by inhibiting enolase enzyme through forming complexes with magnesium • Enolase - requires Mg to act in glycolysis ▪ Clot may occur after few hours ▪ Gray top tubes also include potassium oxalate ▪ Requirement amount of anticoagulant: • 2mg NaF per mL of blood • 2mg K2C2O4 per mL of blood o Hemolyzed: dilutional effect (false ↓) Storage Consideration: o Uncentrifuged clotted blood ▪ Room Temperature (20-25 C): 7 mg/dl/hr decrease ▪ Refrigerator (4 C): 2 mg/dL/hr decrease • Preferred storage method • Glucose Oxidase Method o Most specific enzymatic method o Glucose oxidase - most specific enzyme reacting with only β-D-glucose o D-glucose in blood ▪ α-D-glucose: 34% of total glucose ▪ β-D-glucose: 64% of total glucose o To measure total glucose, mutarotase is added ▪ Mutarotase - convert α-D-glucose to β-D-glucose o Specimens: serum, plasma, CSF, urine, whole blood o Inferences: ▪ False decrease ascorbic acid, uric acid, bilirubin, hemoglobin, tetracycline, glutathione ▪ False increase: bleach o Coupled Enzymatic Reaction ▪ Aka Saifer Gernstenfield method ▪ An enzymatic-colorimetric method (end product is colored and measured using spectrophotometer) ▪ Uses more than one enzyme (glucose oxidase & peroxidase) ▪ Uses reduced chromogen (3-methyl-2benzothiazolinone hydrazone: N,Ndimethylaniline; O-dianisidine) ▪ Utilizes Trinder reaction: CHEMICAL METHODS • Alkaline Copper Reduction Method o Cupric ion is reduced to cuprous ion by the action of a reducing substance like glucose o Principle : ▪ First reaction for Folin Wu, Nelson Somogyi, & Neocuproine o Polarographic Method ▪ Measurement of the degree of oxygen consumption using a pO2 (Clark) electrode ▪ Requires addition of molybdate and iodide or catalase and ethanol ▪ Whole blood samples are not used (false ↑) • Cells consumes oxygen 7 • Hexokinase Method o Reference method for glucose determination o Uses enzymes more accurate than glucose oxidase ▪ G-6-PD - not affected by ascorbic acid & uric acid o Samples: Serum, Plasma, CSF, Urine, Serous fluid o Interferences: Hemoglobin, Drugs, Bilirubin, Lipemia INBORN ERRORS OF CARBOHYDRATE METABOLISM • GLYCOSYLATED HEMOGLOBIN METHODS GLYCOSYLATED HEMOGLOBIN • • • • Specimen: EDTA Whole Blood o Fasting is not required o Only test in CC that require EDTA whole blood Steps: o Hemolyze sample to form hemolysate ▪ Hemolysate - contain lysed RBC solution ▪ 1st reagent - lysing agent ▪ Hemolyzing will release hgb Methods: o Affinity Chromatography ▪ Preferred method of choice ▪ Not affected by HbF, HbS, HbC o Cation-Exchange Chromatography ▪ Temperature-dependent ▪ Affected by HbF, HbS, HbC • HbF can cause false increase • HbS & HbC can cause false decrease o High Performance Liquid Chromatography (HPLC) ▪ All forms of Hgb are measured and quantitated ▪ Separation technique o Electrophoresis o Latex Immunoagglutination Inhibition Methodology Recommended Frequency of Testing for DM Patients (ADA Guidelines) o DM patients with stable glycemic control: twice a year (every 6 months0 o DM patients with unstable glycemic control: quarterly (every 3 months) SELF-MONITORING OF GLUCOSE • Whole Blood Glucose o Point-of-Care Testing (POCT) o Common method for self-monitoring of glucose ▪ Common glucometers o Important in establishing correct insulin amount for next dose o Useful for monitoring hyperglycemia and hypoglycemia among diabetic patient. o Not used for diagnosing hyperglycemia/diabetes ▪ Must be confirmed with lab measurements o Specimen: capillary blood o Recommended Glycemic Target: 80-130 mg/dL, preprandial non-pregnant adult o Frequency of monitoring ▪ Type 1 DM: 3-4x per day ▪ Type 2 DM: frequency is unknown • • • • Galactosemia o Most important melituria ▪ Melituria - presence of any sugar in urine o Congenital deficiency of one of three enzymes involved in galactose metabolism ▪ Galactose-1-phosphate uridyl transferase ▪ Galactokinase ▪ Uridine diphosphate galactose-4-epimerase (GALE) o Causes failure to thrive especially among infants ▪ Infants do not meet standard characteristic/s for specific age groups o Measured in newborn screening o Diagnostic Test: Erythrocyte galactose-1-phosphate uridyl transferase o Laboratory findings: ↑ galactose in blood and urine Essential Fructosuria – fructokinase deficiency Hereditary Fructose Intolerance – fructose-1,6biphosphate aldolase B defect Fructose-1,6-biphosphate Deficiency Glycogen Storage Disease (GSD) o Aka Glycogenoses o Result of inherited deficiencies of enzymes that control the synthesis or breakdown of glycogen o Inherited through autosomal recessive trait except for GSD IXa o Hepatic Glycogenoses / GSD ▪ Includes: GSD I, III, IV, VI, IX ▪ Affects liver and may be associated w/ liver damage ▪ Manifests hepatomegaly, hypoglycemia, and growth retardation o Muscle Glycogenoses ▪ Manifests with exercise intolerance, muscle cramps, fatigue, weakness ▪ Increased: creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), aldolase • Found in skeletal muscle cells GSD Enzyme deficient Notes Type 0 Glycogen synthase Ia Glucose-6-phosphatase • Aka “Von Gierke” • Most common • Hyperlipidemia Ib Glucose-6-phosphatase • translocase II Lysosomal acid alpha • Aka “Pompe” glucosidase IIIa Glycogen debranching • Aka “Cori/Forbes” enzyme IIIb Glycogen debranching enyme IV Glycogen branding • Aka “Andersen” enzyme V Muscle phosphorylase • Aka “McArdle” VI Glycogen • Aka “Hers” phosphorylase VII Phosphofructokinase • Aka “Tarui” IXa Phosphorylase kinase • IXb Phosphorylase • XI Glucose transporter 2 • Aka “Fanconi-Bickle” 8