MLAB 2401: Clinical Chemistry

1
MLAB 2401 Clinical Chemistry

• Renal Assessment

2
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

3
Clinically Significant NPNs
Analyte Plasma Concentration ()
Blood Urea Nitrogen (BUN) 45
Amino Acids 20
Uric Acid 20
Creatinine 5
Creatine 1-5
Ammonia 0.2
4
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

5
BUN Clinical Significance

• Reference range 7-18 mg/dL
• Decreased BUN
• 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

6
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

7
Specimen Requirements BUN

• Plasma
• Serum
• 24-hour Urine
• nonhemolyzed

8
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

9
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

10
Advantages of Creatinine

• Formed at a constant rate
• Readily excreted
• Not reabsorbed
• Not affected by diet

11
Reference Range/Significance Creatinine

• Reference Range

• Significance

• Evaluates renal function
• Follows progress of renal disease
• Increased results
• Renal disease
• Decrease in GFR
• Obstruction in urinary system
• Decreased muscle mass

• Urine
• 0.8-2.0gm/ 24 hour
• Serum
• 0.5-1.5mg/dL

12
Specimen requirements Creatinine

• Plasma
• Serum
• Urine ( 24 hour or random)
• Avoid hemolysis
• Avoid icterus

13
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

14
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

15
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

16
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

17
Creatinine Clearance

• Instructions for urine collection

• Specimen requirements

• 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

• 24-hour urine
• Keep refrigerated
• Serum/Plasma
• Collected during 24-hour urine collection

18
Creatinine clearance -

• Procedure
• Determine creatinine level on serum/plasma - in
  mg/dL
• Determine creatinine level on 24 hour urine
• 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

19
Formula

• Ucr(mg/dL) X V Ur(mL/24 hour)
  X 1.73
• P Cr(mg/dL) X 1440 minutes/ 24 hours
  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

20
Nomogram

1. Left side, find patients height( in feet or
   centimeters)
2. On right side, find patients weight (lbs or kg)
3. Using a straight edge draw a line through the
   points located
4. Read the surface area in square meters, on the
   middle line

21
Reference ranges

• Males
• 97 mL/min- 137 mL/min
• Females
• 88 mL/min-128 ml/min

22
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

23
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

24
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

25
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

26
Clinical Significance Uric Acid

• Gout
• Increased plasma uric acid
• Painful uric acid crystals in joints
• Usually in older males ( gt 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 )

27
Specimen Requirements Uric Acid

• Plasma
• Serum
• Urine
• Serum should be removed from cells ASAP
• Avoid lipemia

28
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 gt Allantoin
  H2O2 CO2
• (Absorbs at 293 nm)
  (Nonabsorbing at 293 nm)

29
Reference Range Uric Acid

• Reference values
• Men 3.5 - 7.2 mg/dL
• Women 2.6 - 6.0 mg/dL

30
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

31
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

32
Specimen Requirements Ammonia

• Whole blood
• EDTA
• Heparin
• Patient should not smoke several hours prior to
  collection, results in contamination

33
Ammonia Methodology

• Glutamate dehydrogenase- enzymatic procedure
• 2 Oxoglutarate NH4 NADPH
  Glutamate NADP H2O
• 2. NADP is measured at 340 nm and it is
  directly proportional to ammonia.

Glutamate dehydrogenase
34
One final note

• Remember the Renal panel
• Albumin
• Glucose
• BUN
• Creatinine
• Calcium
• Chloride
• Potassium
• CO2
• Sodium
• Phosphorus

35
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
  .