(FINAL) CC1 LAB L1

Urea

Clinical Composition of NPNs – The following represents the relative concentration of NPN compounds in the plasma:

• __________: 45–50% (The most abundant NPN)

Amino Acids

Clinical Composition of NPNs – The following represents the relative concentration of NPN compounds in the plasma:

• __________: 25%

Uric Acid

Clinical Composition of NPNs – The following represents the relative concentration of NPN compounds in the plasma:

• __________: 10%

Creatinine

Clinical Composition of NPNs – The following represents the relative concentration of NPN compounds in the plasma:

• __________: 5%

Creatine

Clinical Composition of NPNs – The following represents the relative concentration of NPN compounds in the plasma:

• __________: 1–2%

Ammonia

Clinical Composition of NPNs – The following represents the relative concentration of NPN compounds in the plasma:

• __________: 0.2%

BUN × 2.14

Relationship between BUN and Urea:

• Urea contains nitrogen → BUN reflects part of the urea molecule

• Conversion:

  • Urea (mg/dL) = __________

  • BUN (mg/dL) = —————

    • or Urea ÷ 2.14

  • BUN (mg/dL) → Urea (mmol/L) = —————

Urea × 0.467

Relationship between BUN and Urea:

• Urea contains nitrogen → BUN reflects part of the urea molecule

• Conversion:

  • Urea (mg/dL) = —————

  • BUN (mg/dL) = __________

    • or Urea ÷ 2.14

  • BUN (mg/dL) → Urea (mmol/L) = —————

BUN × 0.357

Relationship between BUN and Urea:

• Urea contains nitrogen → BUN reflects part of the urea molecule

• Conversion:

  • Urea (mg/dL) = —————

  • BUN (mg/dL) = —————

    • or Urea ÷ 2.14

  • BUN (mg/dL) → Urea (mmol/L) = __________

45

Urea

• Approximately ____% of total non-protein nitrogen (NPN)

40–60

Urea

• About ____% of urea is reabsorbed in the renal tubules

Urea cycle (ornithine cycle)

Urea Formation Pathway:

1. Protein → amino acids → deamination → ammonia (NH₃)

2. Ammonia → __________ → urea

3. Urea → transported in blood → kidneys for excretion

Deamination

Urea Formation Pathway:

1. Protein → amino acids → __________ → ammonia (NH₃)

2. Ammonia → urea cycle (ornithine cycle) → urea

3. Urea → transported in blood → kidneys for excretion

Urease

__________ method:

• Reaction: Urea + H₂O —(__________)→ 2NH₃ + CO₂

• Ammonia (NH₃) measurement:

  • Nessler’s reagent

    • Alkaline mercuric iodide solution → yellow color

  • Berthelot reaction

    • NH₃ + phenol + hypochlorite → indophenol (blue color)

  • Ion-selective electrode

    • Measures ammonium ions (NH₄⁺)

Nessler’s reagent

Urease Method

• Reaction: Urea + H₂O —(urease)→ 2NH₃ + CO₂

• Ammonia (NH₃) measurement:

  • __________

    • Alkaline mercuric iodide solution → yellow color

  • Berthelot reaction

    • NH₃ + phenol + hypochlorite → indophenol (blue color)

  • Ion-selective electrode

    • Measures ammonium ions (NH₄⁺)

Berthelot reaction

Urease Method

• Reaction: Urea + H₂O —(urease)→ 2NH₃ + CO₂

• Ammonia (NH₃) measurement:

  • Nessler’s reagent

    • Alkaline mercuric iodide solution → yellow color

  • __________

    • NH₃ + phenol + hypochlorite → indophenol (blue color)

  • Ion-selective electrode

    • Measures ammonium ions (NH₄⁺)

Ion-selective electrode

Urease Method

• Reaction: Urea + H₂O —(urease)→ 2NH₃ + CO₂

• Ammonia (NH₃) measurement:

  • Nessler’s reagent

    • Alkaline mercuric iodide solution → yellow color

  • Berthelot reaction

    • NH₃ + phenol + hypochlorite → indophenol (blue color)

  • __________

    • Measures ammonium ions (NH₄⁺)

Urease–GLDH (Glutamate dehydrogenase)

Coupled __________ Method

• Principle:

  • Urea + 2H₂O —(__________)→ 2NH₄⁺ + CO₃²⁻

  • NH₄⁺ + α-ketoglutarate + NADH —(__________)→ glutamate + H₂O + NAD⁺

• Key Points:

  • Enzymatic, UV, kinetic method

  • 2-point kinetic measurement

  • Measures decrease in absorbance at 340 nm (due to NADH → NAD⁺)

  • Decrease in absorbance is directly proportional to urea (BUN) concentration

340

Coupled Urease–GLDH Method

• Principle:

  • Urea + 2H₂O —(urease)→ 2NH₄⁺ + CO₃²⁻

  • NH₄⁺ + α-ketoglutarate + NADH —(GLDH)→ glutamate + H₂O + NAD⁺

• Key Points:

  • Enzymatic, UV, kinetic method

  • 2-point kinetic measurement

  • Measures decrease in absorbance at ____ nm (due to NADH → NAD⁺)

  • Decrease in absorbance is directly proportional to urea (BUN) concentration

Directly proportional

Coupled Urease–GLDH Method

• Principle:

  • Urea + 2H₂O —(urease)→ 2NH₄⁺ + CO₃²⁻

  • NH₄⁺ + α-ketoglutarate + NADH —(GLDH)→ glutamate + H₂O + NAD⁺

• Key Points:

  • Enzymatic, UV, kinetic method

  • 2-point kinetic measurement

  • Measures decrease in absorbance at 340 nm (due to NADH → NAD⁺)

  • Decrease in absorbance is __________ to urea (BUN) concentration

27, 90

BUN Determination (Urease–GLDH Method Procedure)

• Measurement Conditions:

  • Set spectrophotometer to 340 nm

  • A₁ = ____ seconds

  • A₂ = ____ seconds (exactly)

• Formula:

BUN = (A1-A2)sample / (A1-A2)standard x Cstandard

BUN = (A1-A2)sample / (A1-A2)standard x Cstandard

BUN Determination (Urease–GLDH Method Procedure)

• Measurement Conditions:

  • Set spectrophotometer to 340 nm

  • A₁ = 27 seconds

  • A₂ = 90 seconds (exactly)

• Formula: ____________________

Kinetic UV Assays

• __________:

  • Based on enzymatic reactions (usually urease–GLDH)

  • Measures change in absorbance over time

  • Uses UV light at 340 nm

  • Detects decrease in NADH → NAD⁺

  • Rate of change is proportional to BUN concentration

Dry Chemistry (Dry Density Slides)

• __________:

  • Uses multilayer slides instead of liquid reagents

  • Sample is applied directly onto the slide

  • Reactions occur within layers of the slide

  • Read by an automated analyzer (e.g., Vitros system)

  • Advantages:

    • Less sample preparation

    • Reduced contamination

    • More convenient and automated

Ion-Selective Electrodes (ISE)

• __________:

  • Measures ammonium ions (NH₄⁺) produced from urea

  • Uses a selective electrode that responds to NH₄⁺ concentration

  • Produces an electrical signal proportional to concentration

  • Fast and specific method

Diacetyl (Fearon) Reaction

• __________ — Direct Method

  • Urea reacts with diacetyl (2,3-butanedione) in an acidic medium

  • Forms a yellow chromogen (diazine compound)

  • Reaction:

    • Urea + diacetyl (in acid) → yellow chromogen (diazine)

  • Measurement:

    • Read at 520–540 nm (visible range)

  • Key Points:

    • Direct method → measures urea without enzymatic breakdown

    • Based on color intensity (more color = more urea)

520–540

• Diacetyl (Fearon) Reaction — Direct Method

  • Urea reacts with diacetyl (2,3-butanedione) in an acidic medium

  • Forms a yellow chromogen (diazine compound)

  • Reaction:

    • Urea + diacetyl (in acid) → yellow chromogen (diazine)

  • Measurement:

    • Read at ______ nm (visible range)

  • Key Points:

    • Direct method → measures urea without enzymatic breakdown

    • Based on color intensity (more color = more urea)

1:2

• BUN:Urea ≈ __:__ (For every __ part/s BUN, there are about __ parts urea)

g/L × total volume (L)

• Urine Computation for Urea:

  • g/24 hr urine = ____________________

  • 24-hour urine sample should be diluted 1:20 with NSS (normal saline solution)

1:20

• Urine Computation for Urea:

  • g/24 hr urine = g/L × total volume (L)

  • 24-hour urine sample should be diluted ____ with NSS (normal saline solution)

6–20

Reference Values:

• Plasma/Serum BUN: ____ mg/dL

• Urine Urea (24-hour): 12–20 g/24 hr

12–20

• Reference Values:

  • Plasma/Serum BUN: 6–20 mg/dL

  • Urine Urea (24-hour): ____ g/24 hr

10:1 – 20:1

• BUN/Creatinine Ratio – Used with serum creatinine:

  • Normal = ____

  • Prerenal Azotemia =  > —— (increased)

  • Renal Azotemia = —— (normal or slightly decreased)

20:1

• BUN/Creatinine Ratio – Used with serum creatinine:

  • Normal = ——

  • Prerenal Azotemia =  > ____ (increased)

  • Renal Azotemia = —— (normal or slightly decreased)

10–15:1

• BUN/Creatinine Ratio – Used with serum creatinine:

  • Normal = ——

  • Prerenal Azotemia =  > —— (increased)

  • Renal Azotemia = ____ (normal or slightly decreased)

Isotope dilution mass spectrometry

Detection of characteristic fragments following ionization; quantification using an isotopically labeled compound

Proposed reference method!