CLINICAL CHEMISTRY
CHAPTER 9
NON - PROTEIN NITROGEN
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Introduction
• NPN ( Non - Protein Nitrogen ) is a “funky” term that can be used for
a bunch of different substances that have the element nitrogen in them,
but are not proteins.
• This is a little unusual, because most of the body’s nitrogen is
associated with proteins.
• There are many different unrelated NPNs, but we are only interested in
4 of them:
• Creatinine , Blood Urea Nitrogen ( BUN ) , Uric Acid and Ammonia
• In general, plasma NPNs are increased in renal failure and are
commonly ordered as blood tests to check renal function
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Key Terms
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Allantoin
Ammonia
Azotemia
BUN / Creat Ratio
Creatinine Clearance
Creatine
Creatinine
GFR
Glomerulus
Gout
Hyper ( hypo ) uricemia
NPN
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Pre-renal
Post- renal
Purines
Renal absorption
Renal secretion
Uric acid
Urea
Uremic syndrome
Reyes Syndrome
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Objectives
• List the origin and principle clinical significance of BUN, Creatinine,
Uric Acid and Ammonia
• List the reference ranges for the 4 principle NPNs
• Discuss why creatinine is the most useful NPN to evaluate renal
function
• Calculate Creatinine Clearance
• Discuss the common methodologies used to measure BUN, Creatinine,
Uric Acid and Ammonia
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• General ideas about the NPNs
• Antiquated term when protein – free filtrates were required for testing
• The NPNs were used for evaluating renal function
• The NPNs include about 15 different substances
• Most NPNs are derived from protein or nucleic acid catabolism
• Most important NPNs
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–
–
–
BUN ( Blood Urea Nitrogen )
Creatinine
Uric acid
Ammonia
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• BUN ( Blood Urea Nitrogen )
– Blood Urea Nitrogen = BUN = Urea
– 50% of the NPNs
– Product of protein catabolism which produces ammonia
– Ammonia is very toxic – converted to urea by the liver
– Liver converts ammonia and CO2
Urea
– Filtered by the glomerulus but also reabsorbed by renal tubules ( 40 % )
– Some is lost through the skin and the GI tract ( < 10 % )
– Plasma BUN is affected by
• Renal function
• Dietary protein
• Protein catabolism
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– BUN disease correlations
• Azotemia = Elevated plasma BUN
• Prerenal  BUN ( Not related to renal function )
– Low Blood Pressure ( CHF, Shock, hemorrhage, dehydration )
– Decreased blood flow to kidney = No filtration
– Increased dietary protein or protein catabolism
• Prerenal  BUN ( Not related to renal function )
– Decreased dietary protein
– Increased protein synthesis ( Pregnant women , children )
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– Renal causes of  BUN
• Renal disease with decreased glomerular filtration
– Glomerular nephritis
– Renal failure form Diabetes Mellitus
– Post renal causes of  BUN ( not related to renal function )
• Obstruction of urine flow
– Kidney stones
– Bladder or prostate tumors
– UTIs
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•
BUN / Creatinine Ratio
– Normal BUN / Creatinine ratio is 10 – 20 to 1
– Creatinine is another NPN
– Pre-renal increased BUN / Creat ratio
– BUN is more susceptible to non-renal factors
– Post-renal increased ratio BUN / Creat ratio
– Both BUN and Creat are elevated
– Renal decreased BUN / Creat ratio
– Low dietary protein or severe liver disease
Increased BUN
Normal Creat
Increased BUN
Increased Creat
Decreased BUN
Normal Creat
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– BUN analytical methods
• BUN is an old term, but still in common use
• Specimen : Plasma or serum
• To convert BUN to Urea : BUN x 2.14 = Urea ( mg / dl )
Urease
2 NH4+ + HCO3-
UREA
NH4
+
+ 2-OXOGLUTARATE
GLDH
NADH
GLUTAMATE
NAD
Measure the rate of decreased absorbance at 340 nm
NADH absorbs … NAD does not absorb
Reference range : 10 – 20 mg / dl
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• CREATININE
Liver
Muscles
Muscles
Amino Acids
Creatine
Phosphocreatine
Creatine
Phosphocreatine
Creatinine
Creatinine formed at a constant rate by the muscles as a function of muscle mass
Creatinine is removed from the plasma by glomerular filtration
Creatinine is not secreted or absorbed by the renal tubules
Therefore : Plasma creatinine is a function of glomerular filtration
Unaffected by other factors
It’s a very good test to evaluate renal function
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– Creatinine disease correlations
• Increased plasma creatinine associated with decreased
glomerular filtration ( renal function )
• Glomerular filtration may be 50 % of normal before plasma
creatinine is elevated
• Plasma creatinine is unaffected by diet
• Plasma creatinine is the most common test used to evaluate
renal function
• Plasma creatinine concentrations are very stable from day to
day - If there is a delta check , its very suspicious and must be
investigated
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– Creatinine analytical techniques
• Jaffee Method ( the Classic technique )
Creatinine + Picrate Acid
Colored chromogen
Specimen : Plasma or serum
Elevated bilirubin and hemolysis causes falsely decreased results
Reference range : 0.5 - 1.5 mg / dl
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• URIC ACID
– Breakdown product of purines ( nucleic acid / DNA )
– Purines from cellular breakdown are converted to uric acid by the
liver
– Uric acid is filtered by the glomerulus ( but 98 – 100 % reabsorbed )
– Elevated plasma uric acid can promote formation of solid uric acid
crystals in joints and urine
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– Uric acid diseases
• Gout
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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 )
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– Uric acid analysis
Uricase
Uric acid + O2 + H2O
Allantoin + CO2
+ H2 O2
Uric acid absorbs light @ 293 nm , Allantoin does not.
The rate of decreased absorption is proportional to the uric acid concentration.
Specimen : Plasma or serum
Reference range :
3.5 - 7.2 mg/dl
2.6 - 6.0 mg/dl
Let’s remember
3.0 - 7.0 mg/dl
(males)
(females)
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• AMMONIA
– Produced from the deamaination of amino acids in the muscle and
from bacteria in the GI tract
– Ammonia is very toxic - The liver converts ammonia into urea
– Urea is less toxic and can be removed from the plasma by the
kidneys
– In severe hepatic disease, the liver fails to convert ammonia into
urea, resulting in increased plasma ammonia levels
– Increased plasma ammonia concentrations in :
• Liver failure
• Reye’s Disease
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Ammonia analytical techniques
NH4+ + 2-OXOGLUTARATE + NADPH
L-GLUTAMATE +
NADP+
There is a decreasing absorbance @ 340 nm, proportional to the
ammonia concentration.
Specimen :
EDTA or Heparinized Whole Blood on ice
Must be tested ASAP or plasma frozen
Delayed testing caused false increased values
Reference range : 20 – 60 µg / dl
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• Creatinine Clearance
– Calculated measurement of the rate at which creatinine is removed from
the plasma by the kidneys
– Measurement of glomerular filtration ( renal function )
– A good test of glomerular filtration because
• Creatinine is an endogenous substance ( not affected by diet )
• Creatinine is filtered by the glomerulus, but not secreted or
re-absorbed by the renal tubules
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24 Hour Urine collection
Container.
The volume can be measured
directly off the container.
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– Creatinine Clearance specimens
• 24 hour urine specimen
• Plasma / serum creatinine collected during the urine collection
• 24 Hour Creatinine Clearance Formula
• CREATININE CLEARANCE =
U=
V=
P =
 UV  1.73 



P
A



Creatinine concentration of the 24 hour urine ( mg / dl )
24 hour urine volume ( mls ) per minute - V / 1440 = mls / minute
Plasma creatinine concentration ( mg / dl )
A = Correction factor accounts for differences in body surface area
obtained from a height – weight chart
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Example of a 24 Hour Creatinine Clearance calculation
24 hour urine volume = 1000 mls
24 hour urine creatinine = 20.0 mg / dl
Plasma creatinine
= 5.0 mg / dl
Patients height / weight = 6’00 / 190 lbs
( see pg. 680 )
1000
20.0




UV   1.73 

 1.73 
1440
Creat Cl 




 P  A 
 5.0 
 2.05 
Creat Cl = 2 ml / min
…. Very poor clearance !!!
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• Procedure for 24 Hour Urine Collection
– Have the patient empty his / her bladder ( discard this urine ).
– Note the time . For the next 24 hours, have the patient collect and save all
urine in an appropriate container.
– At the end of the 24 hour period have the patient void one last time into
the urine container. This completes the collection.
– If possible, keep the urine specimen refrigerated.
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– Reference range
• 97 - 137 ml / min ( male)
• 88 - 128 ml / min (female)
• Let’s remember 90 - 130 ml / min
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NPN TOP 10
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Increased Creatinine associated with renal failure
Increased BUN associated with renal failure and protein catabolism
Increased Uric Acid associated with Gout
Increased Ammonia is associated with liver disease
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Creatinine derived from cellular creatine … very constant from day to day
Delta checks on plasma Creatinine must be investigated !!!
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BUN ( Urea ) is derived from protein catabolism
Protein
Ammonia
Urea
Uric Acid is derived from purine( a component of DNA ) catabolism
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Decreased Creatinine Clearance associated with decreased Glomerular
Filtration
 UV  1.73 
Creatinine Clearance  


P
A



Don’t forget to divide V by 1440 !
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Reference Ranges
•
BUN
10 - 20
•
Creatinine
0.5 - 1.5 mg /dl
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Uric Acid
3.0 - 7.0 mg / dl
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Creatinine Clearance
90 - 130 ml / min
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Ammonia
20 - 60
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BUN / Creat Ratio
10 - 20 to 1
mg / dl
ug / dl
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