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Chemistry

Principles

Carbohydrate testing in the lab centers on measuring glucose levels to diagnose and manage diabetes, but also involves assessing other sugars in specific metabolic disorders

  • Glucose Measurement:
    • Principle: Enzymatic methods (glucose oxidase, hexokinase) are most common, measuring the products of glucose oxidation or phosphorylation
    • Methods: Glucose Oxidase, Hexokinase
    • Specimen: Plasma, serum, whole blood (point-of-care)
  • Glucose Tolerance Testing (GTT):
    • Principle: Measures the body’s ability to clear glucose over time after a standard glucose load
    • Method: Serial blood glucose measurements after oral glucose administration
    • Specimen: Plasma or serum at timed intervals
  • Glycated Hemoglobin (HbA1c):
    • Principle: Measures the average blood glucose over the past 2-3 months by assessing the percentage of hemoglobin that is glycated
    • Methods: Immunoassay, HPLC
    • Specimen: Whole blood (EDTA)
  • Urine Glucose:
    • Principle: Detects the presence of glucose in urine, indicating hyperglycemia exceeding the renal threshold
    • Methods: Dipstick, enzymatic methods
    • Specimen: Urine
  • Ketone Measurement:
    • Principle: Detects ketone bodies (acetone, acetoacetate, beta-hydroxybutyrate) in blood or urine, indicating fat breakdown due to insufficient glucose utilization
    • Methods: Immunoassay, enzymatic methods
    • Specimen: Serum, plasma, urine
  • Other Carbohydrates:
    • Principle: Specialized tests for galactose, fructose, etc., typically involve enzymatic or chromatographic methods
    • Methods: Enzymatic assays, chromatography
    • Specimen: Serum, urine

Glucose Measurement

  • Core Concept: Accurate measurement of glucose is central to diagnosing and managing diabetes mellitus

Glucose Oxidase Method (GOD)

  • Principle: Glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide (H2O2). The H2O2 is then reacted with a chromogen in the presence of peroxidase (POD) to produce a colored product, which is measured spectrophotometrically
  • Reactions:
    • Glucose + O2 + H2O –(Glucose Oxidase)–> Gluconic Acid + H2O2
    • H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + H2O
  • Detection: The intensity of the colored product is directly proportional to the glucose concentration in the sample
  • Advantages: Specific for glucose, relatively inexpensive
  • Disadvantages: Susceptible to interference from reducing substances, oxygen availability can affect results

Hexokinase Method (HK)

  • Principle: Hexokinase catalyzes the phosphorylation of glucose by ATP to form glucose-6-phosphate (G6P) and ADP. G6P is then oxidized by glucose-6-phosphate dehydrogenase (G6PD) in the presence of NAD+ to form 6-phosphogluconate and NADH. The NADH produced is measured spectrophotometrically
  • Reactions:
    • Glucose + ATP –(Hexokinase)–> Glucose-6-Phosphate + ADP
    • Glucose-6-Phosphate + NAD+ –(G6PD)–> 6-Phosphogluconate + NADH + H+
  • Detection: The increase in absorbance due to NADH formation is directly proportional to the glucose concentration in the sample
  • Advantages: More accurate and precise than glucose oxidase, less susceptible to interferences
  • Disadvantages: More expensive than glucose oxidase, requires additional enzymes

Glucose Tolerance Testing (GTT)

  • Core Concept: GTT assesses the body’s response to a glucose challenge, providing insight into insulin secretion and sensitivity

Oral Glucose Tolerance Test (OGTT)

  • Principle: A measured dose of glucose is administered orally, and blood glucose levels are measured at specific intervals (e.g., 0, 30, 60, 90, 120 minutes) to assess how the body processes glucose
  • Procedure:
    • Patient Preparation: Overnight fasting (8-12 hours)
    • Glucose Load: Adults typically receive 75 grams of glucose in a liquid solution. Pregnant women may receive a different dose (e.g., 50g for gestational diabetes screening, 100g for diagnostic testing). Children receive 1.75 g of glucose per kg of body weight, up to a maximum of 75 g
    • Blood Collection: Blood samples are collected at timed intervals (e.g., fasting, 30 minutes, 1 hour, 2 hours, 3 hours)
  • Detection: Plasma or serum glucose concentrations are measured at each time point using enzymatic methods (glucose oxidase or hexokinase)
  • Interpretation: Glucose levels are plotted against time to generate a glucose tolerance curve. The shape of the curve and the glucose values at different time points are used to diagnose diabetes, prediabetes, and gestational diabetes
  • Clinical Significance: OGTT is used to diagnose diabetes mellitus, gestational diabetes (during pregnancy), and to assess insulin resistance

Glycated Hemoglobin (HbA1c)

  • Core Concept: HbA1c reflects long-term glycemic control, providing an integrated measure of average blood glucose levels over several weeks

Immunoassay Methods

  • Principle: Antibodies specific to glycated hemoglobin (HbA1c) are used to capture and quantify HbA1c in a blood sample
  • Procedure:
    • Sample Preparation: Whole blood is lysed to release hemoglobin
    • Incubation: The sample is incubated with HbA1c-specific antibodies
    • Detection: The amount of antibody-HbA1c complex formed is measured, and is proportional to the HbA1c concentration
  • Detection Methods:
    • Enzyme-Linked Immunosorbent Assay (ELISA): Enzyme-labeled antibodies are used, and the enzyme activity is measured to quantify the antibody-HbA1c complex
    • Turbidimetric Inhibition Immunoassay (TINIA): Antibody-HbA1c complex formation is measured by the change in turbidity (light scattering)
  • Advantages: Automated, high-throughput, widely available
  • Disadvantages: Susceptible to interferences from hemoglobin variants and other factors affecting red blood cell turnover

High-Performance Liquid Chromatography (HPLC)

  • Principle: HPLC separates different hemoglobin fractions (including HbA1c) based on their chemical properties. The separated fractions are then quantified using spectrophotometric detection
  • Procedure:
    • Sample Preparation: Whole blood is lysed to release hemoglobin
    • Separation: The hemolysate is injected into an HPLC column that separates hemoglobin fractions based on charge or affinity
    • Detection: HbA1c and other hemoglobin fractions are detected by measuring their absorbance at a specific wavelength
  • Advantages: Highly accurate, can detect hemoglobin variants
  • Disadvantages: More complex, requires specialized equipment, more labor-intensive than immunoassay methods

Urine Glucose

  • Core Concept: Urine glucose is a quick indicator of hyperglycemia but is less sensitive than blood glucose measurements

Dipstick Method

  • Principle: A reagent strip impregnated with glucose oxidase, peroxidase, and a chromogen is dipped into the urine sample. Glucose in the urine reacts with glucose oxidase to produce gluconic acid and hydrogen peroxide. The hydrogen peroxide then reacts with the chromogen in the presence of peroxidase to produce a colored product
  • Reactions: Same as Glucose Oxidase Method
  • Detection: The color change on the reagent strip is compared to a color chart to estimate the glucose concentration in the urine
  • Advantages: Simple, rapid, inexpensive
  • Disadvantages: Semi-quantitative, less sensitive than blood glucose measurements, susceptible to interferences from reducing substances and ascorbic acid (Vitamin C)

Enzymatic Methods

  • Principle: Similar to enzymatic methods used for blood glucose measurement (glucose oxidase or hexokinase). The reaction products are measured spectrophotometrically
  • Advantages: More quantitative than dipstick method, more sensitive
  • Disadvantages: More complex, requires instrumentation

Ketone Measurement

  • Core Concept: Ketone testing helps detect ketoacidosis, a serious complication of uncontrolled diabetes

Nitroprusside Reaction (Ketostix)

  • Principle: Acetoacetate and acetone react with nitroprusside in an alkaline medium to produce a purple color
  • Procedure: A reagent strip impregnated with sodium nitroprusside, glycine, and a buffer is dipped into the urine or serum sample
  • Detection: The color change on the reagent strip is compared to a color chart to estimate the ketone concentration
  • Specificity: Primarily detects acetoacetate; acetone reacts less strongly. Beta-hydroxybutyrate is not detected
  • Advantages: Simple, rapid, inexpensive
  • Disadvantages: Semi-quantitative, does not detect beta-hydroxybutyrate (the predominant ketone body in DKA), susceptible to false positives

Beta-Hydroxybutyrate (BHB) Measurement

  • Principle: Beta-hydroxybutyrate dehydrogenase (BHBDH) catalyzes the oxidation of beta-hydroxybutyrate to acetoacetate, with the reduction of NAD+ to NADH. The NADH produced is measured spectrophotometrically
  • Reaction:
    • Beta-Hydroxybutyrate + NAD+ –(BHBDH)–> Acetoacetate + NADH + H+
  • Detection: The increase in absorbance due to NADH formation is directly proportional to the beta-hydroxybutyrate concentration in the sample
  • Advantages: Quantitative, specific for beta-hydroxybutyrate, clinically more relevant in DKA management
  • Disadvantages: More expensive, requires instrumentation

Other Carbohydrates

  • Core Concept: While glucose is the primary focus, specific tests exist for other sugars when investigating metabolic disorders

Galactose Measurement

  • Principle: Galactose oxidase catalyzes the oxidation of galactose to galactonolactone and hydrogen peroxide. The hydrogen peroxide is then reacted with a chromogen in the presence of peroxidase to produce a colored product
  • Reaction:
    • Galactose + O2 + H2O –(Galactose Oxidase)–> Galactonolactone + H2O2
    • H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + H2O
  • Detection: The intensity of the colored product is directly proportional to the galactose concentration in the sample
  • Clinical Significance: Used to diagnose and monitor galactosemia

Fructose Measurement

  • Principle: Fructose can be measured using enzymatic methods involving specific enzymes that convert fructose to measurable products (e.g., using fructokinase)
  • Clinical Significance: Used to diagnose and monitor hereditary fructose intolerance

Chromatography

  • Principle: Chromatography (e.g., HPLC, gas chromatography) can be used to separate and quantify various carbohydrates in a sample based on their physical and chemical properties
  • Clinical Significance: Used for research purposes and in specialized clinical settings to identify and quantify rare sugars and carbohydrates

Key Terms

  • Glucose Oxidase: An enzyme that catalyzes the oxidation of glucose
  • Hexokinase: An enzyme that catalyzes the phosphorylation of glucose
  • Glycated Hemoglobin (HbA1c): Hemoglobin that has glucose attached to it
  • Oral Glucose Tolerance Test (OGTT): A test to assess the body’s response to glucose
  • Ketones: Products of fat breakdown
  • Spectrophotometry: A method to measure the absorbance of light by a solution
  • Chromogen: A substance that produces a colored product
  • Enzymatic Method: A laboratory method that uses enzymes to catalyze a reaction