CARBOHYDRATES

GLUCOSE -primary source of energy for humans especially the brain since it cannot store carbohydrates, it requires a steady supply of glucose to the tissue 1. Embden-Meyerhof pathway ~ Pyruvic acid 2. Hexose Monophosphate Shunt ~ ribose-5-phosphate 3. Glycogenesis ~ glycogen GLYCOLYSIS- the conversion of glucose into pyruvate and lactate GLYCOGENESIS- glucose to glycogen GLYCOGENOLYSIS- glycogen is converted back to glucose GLUCONEOGENESIS- formation of glucose from non carbohydrate sources (TAG-glycerol, skin and muscleslactic acid, protein-amino acids.) -can also be converted to acetyl-coA to enter TCA cycle. -starvation or fasting LIPOGENESIS- Carbohydrates to fatty acid LIPOLYSIS- decomposition of fatty acid

- with hypoglycemic symptoms-shakiness, palpitations, diaphoresis, confusion *injected insulin - inc. insulin low C-peptide. * renal failureC- Peptide and proinsulin because they are degraded in kidney *CIRRHOSIS- hyperinsulinemia *Insulin falsely low in the presence of hemolysis because of insulin degrading enzyme in RBC

2. GLUCAGON
- PROGLUCAGON is synthesized in the pancreatic alpha cells and the the L cells of the distal small bowel. -stimulates glucose production -regulator of hepatic glycogenolysis, gluconeogenesis and ketogenesis *no effect in muscles -GLUCAGONOMAS- rare islet cell tumors that produce excessive glucagon. -characterized by necrotizing migratory erythematous rash, stomatitis, glossitis, weight loss, anemia and mild DM -fasting blood glucagon >120 pg/mL can range from 900-7800 pg/mL -cirrhosis, DM, Cushing's syndrome,pancreatitis, acromegaly, renal insufficiency, familial hyperglucagonemia.

REGULATION 1. INSULIN (hypoglycemic agent)

-secreted by Beta cells of islets of langerhans in response to increased blood glucose level. Mechanism: 1. increasing cell permeability to glucose by biding to receptor on cell surface to increase the uptake of glucose. 2.enhancing the glycogenesis, lipogenesis and protein synthesis 3. inhibit glycogenolysis FUNCTION OF PANCREAS-both exocrine and endocrine. EXOCRINE PANCREAS- produces and secrete amylase -ENDOCRINE PANCREAS secretes four hormones from different cells residing in the islet of langerhans 1. Beta cells - Insulin 2. Alpha cells- Glucagon 3. Delta cells- Somatostatin 4. PP or F cells - Pancreatic polypeptide~ can reduce appetite and food intake Insulin:Glucagon Ratio- regulation of carbohydrate metabolism - Anabolism ~ postprandial state - Catabolism ~ fasting *I:G Ratio is influenced by somatostatin, neural input, intestinal peptides, and concentration of glucose and other metabolites. . PROINSULIN-immediate precursor of insulin.enzymatic removal of C-peptide catalyzed by PC2 and PC2/PC3 (proprotein convertase) - half life atleast 3x than that of insulin. - 10-15% of the biological activity of insulin - FAMILIAL HYPERPROINSULINEMIArare condition caused by mutation in the proinsulin gene. *in Japanese,impaired gluc tolerance or type 2 DM *in caucasian, normal glucose tolerance C-peptide:insulin 5:1 - 15:1 HALF LIFE: C-peptide, proinsulin - 30 mins Insulin- 4-9 minutes -INSULINOMA-insulin and proinsulin,C-peptide (insulin secreting tumors) <50 mg/dl glucose

3. INCRETINS- from intestine

-stimulated by oral nutrients -INCRETIN EFFECT refers to the greater and earlier insulin response to the oral administration of glucose compared with IV glucose

4. SOMATOSTATIN - a tetradecapeptide with SS bond.

-produce in delta cells(5-10% of islet cells) -inhibits insulin and glucagon - inhibits pituitary (GH and thyrotropin) -Gastrointestinal (gastrin, secretin, vasointestinal peptide) -somatostatinoma, small cell lung CA, medullary thyroid CA,and pheochromocytoma -very short lifespan -OCTREOTIDE and LANREOTIDE - long acting somatostatin analog wc bind to the somatostatin receptor subtype 2 (SSTR2) are used to treat neuroendocrine tumors , disorder of pancreas and gastrointestinal tract -OCTREOTIDE SCINTIGRAPHY has also been used for tumor diagnosis, localization and prediction for success of treatment with somatostatin analogs

5. GROWTH HORMONES (SOMATOTROPIN)

-secreted by anterior pituitary. -inhibits glucose uptake by the tissues -stimulate liver glycogenolysis

ADRENOCORTICOTROPIN (ACTH) (CORTICOTROPIN)

6. -secreted by the anterior pitutary -stimulate the release of cortisol

7. CORTISOL
-secreted by adrenal cortex -stimulating gluconeogenesis

8. EPINEPHRINE (ADRENALINE)
-catecholamine secreted by adrenal medulla -increases the blood glucose level by stimulating glycogenolysis and lipolysis -inhibit release of insulin -triggered by physical or emotional stress~causes an immediate increase in the production of glucose with increase heart rate , blood pressure and physiologic effects.

DIABETES MELLITUS
-Most important disease associated with hyperglycemia -heterogenous group of disorder

TYPE 1 DM (formerly IDDM)

-severe form of diabetes characterized with absolute deficiency of insulin because of autoimmune destruction of pancreatic islet of beta cell. -genetic background and environmental changes that might either be chemical or viral -decrease frequency of Human Leukocyte Antigen (HLA) -Associated with DQA and DQB and influenced by DRB genes ANTIBODIES a. islet cell autoantibodies b. insulin autoantibodies c. Glutamic acid decarbolxylase autoantibodies d. Tyrosine phosphatease IA-2 and IA-2B autoantibodies -juvenile diabetes -abrupt onset of fever following a viral of infection and proneness to ketosis. LAB FINDINGS: polyuria hyperglycemia increased serum osmolality and sg ketonemia ketonuria acidosis (acidemia and aciduria) electrolyte imbalance (inc anion gap) Complications: renal cardiac retinal neurological microvascular complications

9. THYROXINE(T4)
-secreted by thyroid gland. -promotes glycogenolysis -can lead to depletion of glycogen stores in the liver -accelerates the rate of glucose absorption from the intestine and may lead to a mildly abnormal, diabetic type of glucose intolerance in the hyperthyroid individuals even though their blood glucose is usually normal

CLINICAL APPLICATIONS
1. HYPERGLYCEMIA- Increase in plasma glucose 2. HYPOGLYCEMIA-Decrease plasma glucose 3. INBORN ERRORS OF CARBOHYDATES METABOLISM normal or decreased plasma glucose concentration, often with excretion of nonglucose reducing sugar in the urine.

HYPERGLYCEMIA
CLASSIFICATION 1. National Diabetes Data Group (1979) a. Insulin-dependent diabetes mellitus (IDDM) b. Non-insulin-dependent diabetes mellitus (NIDDM) c. Impaired glucose tolerance d. Gestational Diabetes Mellitus (GDM) e. Previous abnormality of Glucose tolerance (PotAGT) *>140 mg/dl (7.8 mmol/L) diabetes cut off *Emphasis on glucose tolerance 2. ADA (1997) a. Diabetes type 1 i. Immune mediated ii. Idiopathic b. Diabetes type 2 i. insulin resistance with insulin secretory defects c. Other specific types of diabetes i. Genetic defects of beta cell function ii. Genetic defects in insulin action iii. Diseases of exocrine pancreas iv. Endocrinopathies v. Drug and chemical induced vi. Infections vii Uncommon forms of immunemediated diabetes viii Other genetic syndromes sometimes associated with diabetes d. Gestational diabetes Mellitus (GDM) *emphasis on Fasting plasma glucose (FPG) >126 mg/dl (7.0 mmol/L)

TYPE 2 DM (formerly NIDDM)

-milder -relative deficiency of insulin activity due to insulin resistance (insulin levels maybe normal but there is an insufficient peripheral response to the insulin) and insulin secretory defects. -increased amyloid deposition in their islet cells~ increase glucose intolerance *amyloid is an starchlike protein-carbohydrate complex that can be deposited in tissues, during chronic disease. -has stronger genetic basis because of more frequent familial pattern of occurrence -can be caused by excessive intake of calories leading to weight gain and obesity, ad lack of physical exercise. -no relationship with viruses, no antibodies or HLA association -not prone to ketoacidosis -risk of developing vascular complications and hyperosmolar coma -commonly >40 y. o. -most common 80-90% of patients.

OTHER SPECIFIC TYPES OF DIABETES

1. Genetic defects of beta cell function A. Maturity onset diabetes of youth(MODY)-impaired insulin secretion B. Mitochondrial diabetes mellitus(MtDM) - maternal transmission of diabetes with deafness C. genetic mutation that prevent the convers ion of proinsulin to insulin D. Mutated insulin molecule with poor receptor binding 2. Genetic defects of insulin action A. miscoding for insulin receptor ex: Type A insulin resistance Pedia: leprechaunism, Rabson-medenhall B. Post-receptor signal molecule mutation ex: insulin resistant lipotophic diabetes 3. Pancreatic exocrine disease-decreased/ damaged beta cells -pancreatitis -pancreatic carcinoma -cystic fibrosis -hemochromatosis -fibrocalculous pancreatopathy 4. Endocrine disease- insulin antagonists Cushing's dse (increase cortisol) Glucagonoma (increase Glucagon) Acromegaly (increase GH) Pheochromocytoma (increase epinephrine) Somatostatinoma Aldosteronoma 5. Drug or chemical induced conditions (anti-insulin) impaired beta cell function -dilantin -thiazides -pentamidine -beta adrenergic agonists (poison) Vacor- irreversible B cell destruction 6. Infections Cytomegalovirus Congenital rubella Coxsackie-B4 Adenovirus Mumps 7. Anti-insulin receptor antibody - block insulin binding Type B insulin resistance. -can also be found in patient with SLE *stiff-man syndrome- autoimmune disorder that can develop diabetes 8. Genetic syndrome -down's syndrome -Klinefelter's syndrome -Turner's syndrome -Wolframs syndrome -Malnourish(high circulating fatty acids)

GESTATIONAL DIABETES (GDM) -characterized by onset of diabetes or impaired glucose tolerance (IGT) during pregnancy due to hormonal and metabolic changes. -often accompanied by family history of DM, clinical history of monilial infection, reproductive history of large babies >4000g or infants with congenital anomalies. -should be reclassified following delivery depending on her 6th week plasma glucose level Impaired Glucose Tolerance(IGT) plasma glucose following an oral glucose load (OGTT) not normal yet not sufficiently abnormal to be DM Impaired fasting glucose plasma glucose level following an 8-hour fast that are greater than normal but not sufficiently elevated to be DM. complications of diabetes within 10-15 yrs 1. retinopathy (blindnes) 2. Kidney failure(nephropathy) 3. Neurologic defects (neuropathy) 4. microvascular and macrovascular dse. (heart attacks and stroke) 5. Diminished blood flow to legs and feet due to arteriosclerosis~amputaion, loss of response to normal pressure, minor trauma, susceptibility to infection resulting to gangrene.

LABORATORY DIAGNOSIS
Criteria 1. Symptoms of diabetes plus random plasma glucose concentration 200 mg/dl(11.1 mmol/L) 2. A fasting plasma glucose 126 mg/dl(mmol/L) 3.During OGTT, a 2-hr after oral glucose load plasma glucose 200 mg/dl (11.1mmol/L) *confirmation is not required if the patient is hyper osmolar or ketotic crisis *FPG every 3 years for 45 years if high risk 1.family history of diabetes 2. African, Hispanic, Asian, native americans 3.women with history of large babies. GDM 4.people with HPN 5.people wd low HDL 6.People with High conc of TAG 7. History of IFG or IGT. 1. Casual (Random) Blood Glucose -collected anytime regardless of last meal or time. - 200 mg/dl (11.1mmol/L) 2. Fasting Blood Glucose 10-16 hours fasting : 80-90 mg/dl (4.4-5.0mmol/L) 8 hours fasting <110 mg/dl (6.1mmol/L) - normal 110-125 mg/dl(6.1-6.9mmol/L)- Impaired fasting glucose 126 mg/dl (7.0mmo/L)- DM 3. Urine glucose Renal threshold 160-180 mg/dl(8.9-10 mmol/L) used as screening for DM as insulin therapy guide. 4. Two-hour postprandial Plasma glucose Glucose is measure 2-hours after the patient consume as standard load of glucose (75g of glucose in solution)

5. Oral glucose tolerance test (OGTT) 1. patient should have unlimited physical activity and unrestricted diet containing at least 150g of carbohydrates for 3 days before the test if performed 3. A fasting plasma glucose is collected 4. A glucose dose (conc 25g/dL. and ingested for 5 mins.) 75g - non pregnant 1.75 g/kg body weight -pedia 50g, 100g - pregnant 5. Plasma glucose is measured every 30 minutes for 2 hours (pregnant 3 hrs). Sample should be collected in sodium fluoride preservative, separated and frozen or assayed within 4 hours. For normal patient, the glucose level will drop due to action of insulin while diabetic patient will take longer to decrease glucose conc. 140 mg/dl (7.8) on 2hr - Normal 140-199 mg/dl (7.8-11)- impaired glucose tolerance 200 mg/dl (11.1) on 2hr confirmed with RBS or FBS - DM For pregnant: IF two are met = GDM 1.) FBS 105 mg/dl(5.8) 2.) 1-hr 190 mg/dl (10.6) 3.) 2-hr 165 mg/dl (9.2) 4.) 3-hr 145 mg/dl (8.1) 6. Screening for Gestational DM -should be performed bet 24-28 weeks of gestation. -no fasting -50 g of glucose , collect 1 hr sample 140 mg/dl mandates complete 3hr OGTT 7. Intravenous Glucose tolerance test -for poor absorption of orally administer and intolerability oral carbohydrate load -0.5g/kg of body administer IV and glucose determined every 10mins for 1 hr. 8. Glycated/ Glycosylated Hemoglobin (HBA1C)/ "fast hemoglobin"/ glycohemoglobin Hgb A(95-97%) , Hgb A2 (2.5%), HgbF (0.5%) minor hgb component of HgB A (80%) -provides an index of patient average blood glucose over 2-3month period -useful for determining compliance with therapy. -advanage over OGTT, requires only 1 sample -should be 7% HBA1c 9. Fructosamine -glucose-protein ketoamine linkages -due to the fact that all protein can be glycated and albumin is the most abundant protein in serum.thus, measure of fructosamine is largely a measure of glycated albumin. -reflection of short term glucose control (2-3 weeks) -colorimetric procedure - ability of fructosamine linkage to reduce nitroblue tetrazolium -respond more quickly to changes of glucose control and not affected by abnormal hemoglobin and rapid hemoglobin turnover

Sample: 1.) random urinary albumin to creatinine ratio 2.)24-hour urinary albumin concentration 3.) 4-hour urinary albumin concentration 4.) first morning void urinary albumin concentration Urine dipstick (550 mg/day) proteinuria- Irreversible. 11. Serum and Urine Ketone tests -indicator of degree of ketoacidosis (type 1 DM) nitropruside with or without glycerin- detect acetoacetic acid and acetone or acetoacetic acid alone. B-hydroxybutyrate dehydrogenase- detect Bhydroxybutyric acid in acute ketoacidosis 12. Insulin testing-to discriminate DM1 form DM2 in patient who are progressing from 2 to 1. 13. C-peptide- used to detect endogenous insulin production in patients being treated with exogenous insulin

HYPOGLYCEMIA - Low plasma glucose

<50 mg/dl (2.8) *lower limit: 35 mg/dl (1.9)in healthy premenopausal women who had fasted for 24 hours. CLASSIFICATION 1. Reactive hypoglycemia- caused by excessive administration of insulin or other hypoglycemic agent ( factitious hypoglycemia) or by the reduction in gluconeogenesis as a result of ethanol ingestion. -may occur several hours after a meal (post prandial hypoglycemia)in individuals who have had gastrointestinal surgery or in mild diabetics. 2 Fasting/ Spontaneous hypoglycemia -can be caused by excessive insulin secreted by insulin producing pancreatic islet cell carcinoma(insulinomas), nonpancreatic tumor which produce substances with insulin-like activity, hepatic dysfunction, glucocorticoid deficiency, sepsis, or depleted glycogen stores.

*rapid fall in plasma glucose triggers the release of epinephrine (sweating, weakness, shakiness, trembling, nausea, hunger, rapid pulse, lightheadedness and epigastric discomfort) *gradual fall of glucose to less than 20 or 30 mg/dl (1.1 or 1.7 mmol/L) cause impairment to CNS since the brain depends on an adequate supply of glucose for its energy. symptoms: neuroglycopenia, headache, confusion, lethargy, seizure and unconsciousness~ irreversable brain damage or death. Infants abnormal <30 mg/dl (1.7)- term infant <20 mg/dl (1.1)- premature Condition causing hypoglycemia in neonates and children -maternal diabetes or eclampsia -prematurity -polycytemia -respiratory distress syndrome -glycogen storage disorders -gluconeogenic

10. Urine microalbumin test -early REVERSIBLE stage of diabetic nephropathy -microalbuminuria - Low concentration of albumin (30300mg/day) seen in urine

LABORATORY DIAGNOSIS 1. Plasma glucose -measure every 4 hours -Abnormal low glucose after 12 hrs fasting~fasting hypoglycemia -No glycemia demostrated after 48hrs -hypoglycemia triggered by food intake -> get RBS -> if normal then it is not hypoglycemia 2. Five-Hour Glucose Tolerance Test (5-h GTT) -standard test to diagnose post prandial hypoglycemia but it is not sensitive and specific -glucose is administered orally, draw blood every 30 mins for 5 hours or when pc develop hypoglycemic symptoms - < 50 mg/dl (2.8) may suggest postprandial hypoglycemia. 3. Insulin levels -Radioimmunoassay (RIA) -increase insulin with decrease glucose suggestive of insulin-producing pancreatic islet cell tumor -after 72 hrs fasting insulin:glucose (<0.30 U/mL:mg/dl) 4. C-peptide a. to differentiate whether the source of hypoglyemia is endogenous or exogenous. b. useful for insulin treated diabetic who usually develop anti-insulin antibodies that will interfere with insulin determination. Thus C-peptide can be measured instead., to evaluate residual beta cell function secretory function c. used to indicate the residual pancreatic tissue in postpancreatectomy. C-peptide suggest recurrence or metastases 5. Insulin tolerance test- to evaluate patients with resistance to administered insulin or with certain other endocrine disorders. -FBS, IV insulin, measure glucose several intervals for 2 hours after insulin admin. Normal: after 30m- dec glu about half the FBS after 90-120m - return to normal *resistance to insulin (adrenal cortical hyperperfunction, acromegaly, diabetes) - slight or delayed dec in glucose *Hypofunction of anterior pituitary or adrenal cortex (dwarfism or addison's dse)-blood glucose decreases normally in response with insulin but delayed rise or does not occur at all in the subsequent hrs. 6 Tolbutamide tolerance test -use to differentiate insulinomas from other hyperinsulinemic states -FBS, IV tolbutamide measure glucose at interval for 2 hrs. -Tolbutamide(1-butyl-3-(p-tolysulfonyl)urea) stimulates pancreas to produce and secrete insulin. -response similar to Insulin tolerance test.

INBORN ERROR OF METABOLISM

1.GLYCOGEN STORAGE DISEASE deficiency of one or
more enzymes involved in the metabolism of glycogen. TYPE I (von Gierke's) ENZYME DEFICIENCY Glucose-6phosphatase CLIN SYMPTOMS Severe hepatomegaly and hypoglycemia, lactic acidosis hyperlipidemia , failure to thrive Infantcardiomegaly,muscl e weakness, early death Adult- muscle weakness Hepatomegaly, muscle weakness, hypoglycemia Hepatomegaly, cirrhosis, failure to thrive, early death

II (Pompe's)

- 1,4-glucosidase

III (Cori's)

Amylo-1,6glucosidase (debrancher) -1,4-glucan: -1,4-glucan,6glucosyl-transferase (brancher) Muscle phosphorylase

IV (Anderson's )

V (McArdle's)

Muscle cramps after exercise, myoglobinuria in half the patients Hepatomegaly, mild clinical course Muscle cramps after exercise, myoglobinuria in some patients Spasticity, decelebration, high urinary catecholamine, death in infancy Hepatomegaly, increased liver glycogen concentrations Hepatomegaly only

VI (Her's) VII (Tauri's)

Hepatic phosphorylase Muscle phosphofructokinas e

VIII

Adenyl Kinase

IX

Hepatic phosphorylase b kinase

Cyclic AMPdependent kinase Epinephrine tolerance- use to evaluate this form of glycogen storage dse.

2. GALACTOSEMIA- characterize by a deficiency or

absence of one of the three enzymes involved in the metabolism of galactose. galactokinase galactose-1-phosphate uridyl transferase Uridyl diphosphate glucose-4-epimerase Dx: diagnosis of galactose in urine or serum confirmatory test : finding 1 -phosphate uridyl transferase in RBC Screening program: measuring activity of transferase in RBC

3. DISORDER OF FRUTOSE METABOLISM A. HEREDITARY FRUCTOSE INTOLERANCE- deficiency of fructose-1-phosphate aldolase B. Fructose-1,6-diphosphatase deficiency C. ESSENTIAL FRUCTOSURIA-deficiency of hepatic fructokinase 4. Mucopolysaccharidoses- mucopolysaccharide storage diseases caused by deficiency of one or more lysosomal enzymes. 3 classes of mucopolysaccharide 1. dermatan sulfate 2. heparan sulfate 3. Keratan sulfate Hurler syndrome- prototype for all mucopolysaccharide storage diseases CEREBROSPINAL FLUID (CSF) -Glucose is 60-70% of that in the plasma, or 40 to 70 mg/dl (2.2 to 3.9) -2-3 hours for a change in plasma glucose conc to reflect on the CSF due to transport of glucose across the bloodCSF barrier -DECREASE - bacterial, tuberculous or fungal meningitis, systemic hypoglycemia (<40 mg/dl or 2.2mmol/L) due to: 1. Impaired transport of glucose from blood to CSF 2. increase glucose utilization by the brain tissue 3. increased glucose utilization by bacteria, leukocytes and neoplastic cells in CSF. ANALYTICAL PROCEDURES -Serum, plasma, Whole blood. * Whole blood has 10-15% higher glucose than serum/plasma ADVANTAGE OF USING SERUM/PLASMA 1. more suitable for automated method 2.hematocrit does not interfere with serum or plasma glucose esp when 3. Erythrocytes also contain nonglucose reducing substances that could interfere with measurement of glucose when using reducing agent. 4. eliminates the needs for preservative ANTIGLYCOLYTICS AGENT: 1. Fluoride- inh enolase and fructose-6-phosphatease 2. Iodoacetate- inh phosphoglyceraldehyde dehydrgenase *24 hrs at RT/ antiglycolytic agent without anticoagulant, separated serum/plasma from cells 8 hours at RT 72 hours at ref VENOUS VS CAPILLARY Fasting- capillary is 3mg/dl (0.2 mmol/L) 15-19 mins afterglucse load- capillary is 32 mg/dl (1.8mmol/L) higher *sample should never be collected from the IV site METHODS I. CHEMICAL METHOD 1. OXIDATION REDUCTION METHODS a. Alkaline Copper methods i. Folin Wu method ii. Nelson Sumogyi method iii. Neocuproine method iv. Benedict's method (modification of folin Wu)

2.

b. Alkaline Ferric Reduction Method (Hagedorn Jensen) CONDENSATION METHOD (Dubowski mtd) O-toluidine

II. ENZYMATIC METHODS 1.GLUCOSE OXIDASE METHOD a. Colorimetric glucose oxidase method b. Polarographic Glucose Oxidase 2. HEXOKINASE METHOD 3. GLUCOSE DEHYDROGENASE 4.DEXTROSTICS (CELLULAR STRIP) 5. GLYCOSYLATED HEMOGLOBIN (HBA1C) 6. FRUCTOSAMINE

A. OXIDATION REDUCTION METHOD 1. ALKALINE COPPER REDUCTION METHODS Principle: Reduction of cupric ions to cuprous ions forming cuprous oxide in hot alkaline solution by glucose Alkaline Copper Tartrate Cuprous ions

a. Folin Wu Method Cuprous ions + phosphomolybdate Phosphomolybdic acid/ phosphomolymbdenum Blue b. Nelson Somogyi Method Cuprous ions + Arsenomolybdate Arsenomolybdic acid/ Arsenomolybdenum Blue c. Neocuproine Method (2,9 Dimethyl 1,10 Phenantorline Hydrochloride) Cuprous ions + Neocuproine Cuprous- Neocuproine Complex (yellow or yellow orange) d. Benedict's method (Modification of Folin Wu) -used for the detection and quantitation of reducing substances in body fluids like blood and urine - Use citrate or tartrate as stabilizing agent 2. ALKALINE FERRIC REDUCTION METHOD (HAGEDORN JENSEN) -reduction of yellow ferricyanide to a colorless ferrocyanide by glucose (Inverse colorimetry) B. CONDENSATION METHOD Ortho-toluidine (Dubowski method) glucose + Aromatic amines glycosamine + schiff base

II. ENZYMATIC 1. Glucose oxidase method (Enzymatic colorimetric) -measures the B-D glucose -also measures CSF glucose a. Colorimetric glucose oxidase mtd (Saifer Gernstenfield) Glucose + Gluconic acid + Oxidized Chromogenic subs. + H20

+ Chromogenic subs

b. Polarographic Glucose oxidase -The enzymatic conversion of glucose is quantitated by the consumption of oxygen on an oxygen- sensing electrode -measures the rate of oxygen consumption which is proportional to the glucose concentration. -Glucose oxidase in the reagent catalyzes the oxidation of glucose by oxygen first under first order conditions, forming hydrogen peroxide. -the hydrogen peroxide is prevented from reforming oxygen by adding molybdate, iodide , catalase and ethanol Glucose + O2 H2O2 + C2H5OH H2O2 + 2H + 2I Gluconic acid + h2O CH3CHO + 2H2O I2 + 2H2O

2. Hexokinase Method -Most specific glucose method reference method -Plasma collected using Heparin , EDTA, fluoride, oxalate or citrate maybe use for this test. -Other samples: urine, CSF and serous fluids a. Glucose + ATP Glucose-6-phosphate + ADP b. Glucose-6-phosphate + NADP (oxidized) Glucose-6-phosphate dehydrogenase 6- Phosphoglucolactone + NADPH 3. Glucose Dehydrogenase Method -The amount of NADH generated is proportional to the glucose concentration -It provides result in close agreement with hexokinase procedures Mutarotase is also added to shorten time necessary to reach equilibrium Glucose D-Glucose-d-lactone

4. Dextrostics -important in establishing correct insulin amount for next dose -Effective in reducing the rate of development of diabetic complications 5. Glycosylated Hemoglobin -also called glycated hemoglobin refects ave glucose level over 2-3 months 6. Fructosamine -also called glycosylated albumin/ plasma protein ketoamine -refection of short term glucose 2-3 weeks -monitoring diabetic individuals with Chronic hemolytic anemias and hemoglobin variants (Hb S or Hb C) decreased RBC lifespan -should not be use in presence of low albumin (<30 g/L)