MLAB 2401: Clinical Chemistry
Renal Assessment
Nonprotein Nitrogen Compounds
• What are they?
– Products from the catabolism of proteins and
nucleic acids
– Consist of a molecule that contains nitrogen but
are not part of a protein
– Useful to evaluate renal function
Clinically Significant NPN’s
Analyte
Plasma Concentration (%)
Blood Urea Nitrogen (BUN)
45
Amino Acids
20
Uric Acid
20
Creatinine
5
Creatine
1-5
Ammonia
0.2
BUN
• Blood Urea Nitrogen
– Urea is the nitrogenous end-product of protein &
AA metabolism.
– Urea is formed in the liver when ammonia (NH3) is
removed and combined with CO2.
– Rises quickly as compared to creatinine
– Majority excreted in urine
– Most widely used screening test of kidney
function
BUN: Clinical Significance
• Reference range 7-18 mg/dL
• Decreased BUN
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Late pregnancy
Decreased protein intake
Severe liver disease
Overhydration
• Increased BUN
– Azotemia
• Occurs when BUN concentration exceeds 20 mg/dL
• Not always due to kidney malfunction
BUN / Creatinine Ratio
– Normal
 BUN / Creatinine ratio is 12 – 20 to 1
– Pre-renal Azotemia
 Increased BUN due to non-renal causes
 Congestive heart failure, high protein diets, dehydration
 Increased Ratio- BUN is high/ creatinine is normal
– Renal Azotemia
 Disease directly affects nephron
 Glomerulonephritis, Nephrotic syndrome, uremia, etc.
 Normal Ratio- both BUN and creatinine are proportionally elevated
– Post-renal Azotemia
 Occurs after urine has left the kidney- due to obstruction
 Increased Ratio- BUN is high
 Plasma creatinine also elevated
Specimen Requirements: BUN
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•
•
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Plasma
Serum
24-hour Urine
nonhemolyzed
BUN: Methodology
• Kjeldahl – a classical method for determining urea
concentration by measuring the amount of nitrogen
present
• Berthelot reaction - Good manual method - that measures
ammonia
– Uses an enzyme (urease ) to split off the ammonia
• Diacetyl monoxide ( or monoxime)
– Popular method but not well suited for manual
methods
• because ➵ Uses strong acids and oxidizing
chemicals
Creatinine/Creatine
• Creatinine is formed from creatine and creatine
phosphate in muscle
• Metabolic product cleared entirely by glomerular
filtration
• Not reabsorbed
• In order to see increased creatinine in serum,
50% kidney function is lost
• Creatinine levels are affected by muscle mass,
creatine turnover, and renal function
Advantages of Creatinine
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•
•
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Formed at a constant rate
Readily excreted
Not reabsorbed
Not affected by diet
Reference Range/Significance:
Creatinine
Significance
• Evaluates renal function
• Follows progress of renal
disease
Reference Range
• Urine
– 0.8-2.0gm/ 24 hour
• Serum
• Increased results
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–
–
–
Renal disease
Decrease in GFR
Obstruction in urinary system
Decreased muscle mass
– 0.5-1.5mg/dL
Specimen requirements: Creatinine
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•
•
•
•
Plasma
Serum
Urine ( 24 hour or random)
Avoid hemolysis
Avoid icterus
Creatinine: Methodology
• Jaffe reaction
– basic reaction for creatinine
– Kinetic
• Principle: Protein-free filtrate(serum/urine) mixed with
alkaline picrate solution forms a yellow-orange complex
of creatinine picrate which absorbs light at 520 nm,
proportional to the amount of creatinine present
• Issues
– Subject to interferences from proteins, glucose, uric
acid, medications and others
– Enzymatic
• New technology involving coupled reactions
Clearance Measurements
• Evaluation of renal function relies on waste
product measurement, specifically the urea
and creatinine
• Renal failure must be severe, where only 20%
of the nephron is functioning before
concentrations of the waste products increase
in the blood
• The rate that creatinine and urea are cleared
from the body is termed clearance
Clearance
• Definition
– Volume of plasma from which a measured amount
of substance can be completely eliminated into
urine per unit of time
– Expressed in milliliters per minute
• Function
– Estimate the rate of glomerular filtration
Creatinine Clearance
• Used to estimate GFR ( glomerular filtration
rate)
• Most sensitive measure of kidney function
• Mathematical derivation taking into effect the
serum creatinine concentration to the urine
creatinine concentration over a 24- hour
period
Creatinine Clearance
Specimen requirements
• 24-hour urine
– Keep refrigerated
• Serum/Plasma
– Collected during 24-hour
urine collection
Instructions for urine collection
• Empty bladder, discard
urine, note exact time
• Collect, save and pool all
urine produced in the next
24-hours.
• Exactly 24 hours from start
time, empty bladder and
add this sample to the
collection
Creatinine clearance Procedure
– Determine creatinine level on serum/plasma - in
mg/dL
– Determine creatinine level on 24 hour urine
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•
•
•
measure 24 hr. urine vol. in mL, take a aliquot
make a dilution (usually X 200)
run procedure as for serum
multiply results X dilution factor
– Plug results into formula
Formula
Ucr(mg/dL) X V Ur(mL/24 hour)
X
P Cr(mg/dL) X 1440 minutes/ 24 hours
1.73
A
• U cr= urine creatinine
• P cr= serum creatinine
• 1.73= normalization factor for body surface
area in square meters
• A= actual body surface area
Nomogram
1.
2.
3.
4.
Left side, find patient’s height( in feet or centimeters)
On right side, find patient’s weight (lbs or kg)
Using a straight edge draw a line through the points located
Read the surface area in square meters, on the middle line
Reference ranges
• Males
 97 mL/min- 137 mL/min
• Females
 88 mL/min-128 ml/min
Creatinine Clearance Exercise
• Female Patient: 5'6“ & 130 lbs.
– Urine Creatinine – 98 mg/dL
– Serum Creatinine – 0.9 mg/dL
– 24 Hour Urine Volume – 1,200 mL
– Set up calculation
Drawbacks of Creatinine Clearance
• Overestimates the GFR by 10-20%
• Timing of serum/urine collection for accurate
analysis
• Patients/Health care workers must follow
detailed instructions for proper collection
New Ways to Evaluate eGFR
• Estimates GFR from serum creatinine
• Patients age, sex, weight, or race included in
the equation
• Common equation used include:
– Modification of Diet in Renal Disease (MDRD)
– Cockcroft-Gault
– CKD-EPI
Uric acid
• Final breakdown product of nucleic acid catabolism - from
both the food we eat, and breakdown of body cells.
• Uric acid is filtered by the glomerulus, majority reabsorbed
• Roles
– Assess inherited purine disorders
– Confirm diagnosis and treatment of gout
– Assist in diagnosis of renal calculi
– Prevent uric acid nephropathy during chemotherapy
– Detect kidney dysfunction
Clinical Significance: Uric Acid
•Gout
–Increased plasma uric acid
–Painful uric acid crystals in joints
–Usually in older males ( > 30 years-old )
–Associated with alcohol consumption
–Uric acid may also form kidney stones
• Other causes of
increased uric acid
– Leukemias and
lymphomas
» (  DNA catabolism )
– Megaloblastic anemias
» (  DNA catabolism )
– Renal disease ( but not
very specific )
Specimen Requirements: Uric Acid
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•
•
•
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Plasma
Serum
Urine
Serum should be removed from cells ASAP
Avoid lipemia
Uric Acid: Methodology
1. Phosphotungstic Acid Reduction — This is the classical chemical method
for uric acid determination. In this reaction, urate reduces
phosphotungstic acid to a blue phosphotungstate complex, which is
measured spectrophotometrically.
2. Uricase Method — An added enzyme, uricase, catalyzes the oxidation of
urate to allantoin, H2O2, and CO2. The serum urate / uric acid may be
determined by measuring the absorbance at 293 nm before and after
treatment with uricase. (Uricase breaks down uric acid.)
Uric acid + 2H2O + O2 Uricase > Allantoin + H2O2 + CO2
(Absorbs at 293 nm)
(Nonabsorbing at 293 nm)
Reference Range: Uric Acid
• Reference values
 Men 3.5 - 7.2 mg/dL
 Women 2.6 - 6.0 mg/dL
Other Screening Test for Renal Disease
• Urinalysis
– Routine urinalysis good indicator of renal disease
• Microalbumin
– Albumin is another sign of renal disease
– Usually performed on a random urine
Ammonia
• Formed from the breakdown of amino acids
and bacterial metabolism
• Metabolized by the liver
• Increases due to renal failure or liver disease
are toxic to the CNS
Specimen Requirements: Ammonia
• Whole blood
– EDTA
– Heparin
– Patient should not smoke several hours prior to
collection, results in contamination
Ammonia: Methodology
1. Glutamate dehydrogenase- enzymatic procedure
2 Oxoglutarate + NH4+ + NADPH
Glutamate
dehydrogenase
Glutamate + NADP+ + H2O
2. NADP + is measured at 340 nm and it is directly proportional to ammonia.
One final note…
• Remember the Renal panel
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Albumin
Glucose
BUN
Creatinine
Calcium
Chloride
Potassium
CO2
Sodium
Phosphorus
References
• Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques,
principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams &
Wilkins.
• Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper
Saddle River: Pearson .
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