Glucose, quantitative determination

Hormonal regulation of blood glucose

Methods

• Chemical

• Enzymatic

CHEMICAL METHODS

• Oxidation - Reduction Methods – Folin – Wu– Somogyi - Nelson

• Condansation Methods– Orto - Toluidine

Benedict reaction

Folin - Wu

Glucose + CuGlucose + Cu2+2+ >>>Gloconate + >>>Gloconate + CuCu++

pH > 7 , heatpH > 7 , heat

CuCu++ + phosphomolybdate + phosphomolybdate

Blue molybdenium complexBlue molybdenium complex λλ = 660 nm = 660 nm

Somogyi - Nelson

Glucose + CuGlucose + Cu2+2+ >>> >>> Gloconate + CuGloconate + Cu++

pH > 7 , heatpH > 7 , heat

CuCu++ + arsenomolybdate >>> + arsenomolybdate >>> blue molybdenium complex blue molybdenium complex

λλ = 660 nm = 660 nm

O - Toluidine Method

o - Toluidineo - Toluidine + Glucose + Glucose >>> >>> GlycosylamineGlycosylamine

pH < 7 , heatpH < 7 , heat

Schiff BaseSchiff Base

λλ = 630 nm color complex = 630 nm color complex

CH3

NH2

Interference

• Bilirobin• Galactose• Mannose• Hemoglobin

Enzymatic Methods

• Glucose Oxidase

• Hexokinase

• Glucose dehydrogenase

Glucose Oxidaseαα -D-Glucose -D-Glucose ββ-D-Glucose-D-Glucose

OO22 glucose oxidaseglucose oxidase

Gluconic acid + Gluconic acid + HH22OO22 PeroxidasePeroxidase

HH22OO22 + chromogen + chromogen Color complexColor complex (o-dianisidine)(o-dianisidine) + H + H22O O (phenylamine)(phenylamine) λλ = 520 nm = 520 nm

Mutarotase

• Glucose oxidase Inhibited by– High concentrations of uric acid, ascorbic acid,

bilirubin, glutathione, creatinine. L-cysteine, L-dopa. Dopamine, methyldopa and citric acid

Hexokinase method

Glucose dehydrogenase method

Clinical Significance

• F.B.S (Fasting Blood Suger)• Fasting 12 - 14 hours• Normal Range 70 - 110 mg/dL• Decrease

– Hypoglycemia

• Increase – Hyperglycemia

Criteria for the diagnosis of diabetes mellitus

Diagnosis of Pre-Diabetes and Diabetes Mellitus

Hyperglycemia

• FBS > 140 mg/dL• Primary (Diabetes Mellitus)

– IDDM (type I)– NIDDM (type II)

• Absolute deficiency of insulin– Make up about 10% of all patients with D.M.

• IDDM persent at an early age (usually before 30)

• Clinical signs :– Blood glucose– Insulin (decreased)– Ketosis – Loss of body weight

Insulin Dependent Diabetes Mellitus (IDDM)

Noninsulin Dependent Diabetes Mellitus (NIDDM)

• Deficiency of insulin receptorDeficiency of insulin receptor• Patients are commonly obesePatients are commonly obese• NIDDM is the most common from of D.M. ( 80-NIDDM is the most common from of D.M. ( 80-

90%)90%)• Usually first present at an age over 40Usually first present at an age over 40• Clinical signsClinical signs

– Blood glucose– Insulin– Osmotic diuresis – Dehydration

Characteristics of Type 1 and Type 2 Diabetes

Response of plasma insulin to glucose stimulation

Secondary hyperglycemia

• Pancratectomy

• Acromegaly

• Cushing’s syndrome

• Glucagonoma

Hypoglycemia

• Adult < 45 - 50 mg/dL in fasting

• Infant – Preterm < 25 mg/dL– Fullterm < 35 mg/dL

Causes of hypoglycemia

• Insulinoma (islet cell tumors)• Glycogen Storage Diseases (GSD)• Addison’s disease

OGTT

Fasting 80 mg/dL0.5 155 mg/dL1 165 mg/dL2 140 mg/dL3 80 mg/dL

Oral Glucose Tolerance Tests (OGTT)

Screening and diagnosis of gestational diabetes mellitus

OGTT curve

2hpp

• 2hpp (two hours postprandial plasma glucose) – To screen for diabetes mellitus – To monitor glucose control

Glycated hemoglubin

• HbA1c

• an index of average blood glucose levels over the past 2-4 months.

• People without diabetes have HbA1c levels between 4-6%.

• Measure HbA1C levels every 3-6 months to monitor glycemic control

Blank Standard Test

Water 0.05 ml - -

Standard 100 mg/dL

- 0.05 ml -

Test - - 0.05 ml

Reagent 3 ml 3 ml 3 ml

• Put 10 minute in boil water bath

• read at 630 nm

• Ct = At / Ast * Cst