1. CLINICAL CHEMISTRY CHAPTER 9
NON - PROTEIN NITROGEN

2. 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

3. Key Terms Pre-renal Post- renal Purines Renal absorption
Allantoin
Ammonia
Azotemia
BUN / Creat Ratio
Creatinine Clearance
Creatine
Creatinine
GFR
Glomerulus
Gout
Hyper ( hypo ) uricemia
NPN
Pre-renal
Post- renal
Purines
Renal absorption
Renal secretion
Uric acid
Urea
Uremic syndrome
Reyes Syndrome

4. 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

5. 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
BUN ( Blood Urea Nitrogen )
Creatinine
Uric acid
Ammonia

6. Urea 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
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
Urea

7. 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 )

8. Renal disease with decreased glomerular filtration
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

9. 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

10. BUN analytical methods BUN is an old term, but still in common use
Specimen : Plasma or serum
To convert BUN to Urea : BUN x = Urea ( mg / dl )
Urease
2 NH HCO3-
UREA
GLDH
NH OXOGLUTARATE
GLUTAMATE
NADH
NAD
Measure the rate of decreased absorbance at 340 nm
NADH absorbs … NAD does not absorb
Reference range : – 20 mg / dl

11. Liver Amino Acids Creatine Muscles Creatine Phosphocreatine
CREATININE
Liver Amino Acids Creatine
Muscles Creatine Phosphocreatine
Muscles 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

12. 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

13. 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 : mg / dl

14. 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

15. Uric acid diseases 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 )

16. + Uric acid analysis Uricase Uric acid + O2 + H2O Allantoin + CO2 H2O2
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 : mg/dl (males)
mg/dl (females)
Let’s remember mg/dl

17. Ammonia is very toxic - The liver converts ammonia into urea
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

18. Ammonia analytical techniques
NH OXOGLUTARATE + NADPH L-GLUTAMATE +
NADP+
There is a decreasing 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

19. Creatinine is an endogenous substance ( not affected by diet )
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

20. 24 Hour Urine collection
Container.
The volume can be measured
directly off the container.

21. Creatinine Clearance specimens
24 hour urine specimen
Plasma / serum creatinine collected during the urine collection
24 Hour Creatinine Clearance Formula
CREATININE CLEARANCE =
U = Creatinine concentration of the 24 hour urine ( mg / dl )
V = hour urine volume ( mls ) per minute V / 1440 = mls / minute
P = Plasma creatinine concentration ( mg / dl )
A = Correction factor accounts for differences in body surface area
obtained from a height – weight chart

22. Example of a 24 Hour Creatinine Clearance calculation
24 hour urine volume = mls
24 hour urine creatinine = mg / dl
Plasma creatinine = mg / dl
Patients height / weight = 6’00 / lbs ( see pg. 680 )
Creat Cl = ml / min …. Very poor clearance !!!

23. 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.

24. Reference range
ml / min ( male)
ml / min (female)
Let’s remember ml / min

25. NPN TOP 10 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
Creatinine derived from cellular creatine … very constant from day to day
Delta checks on plasma Creatinine must be investigated !!!
BUN ( Urea ) is derived from protein catabolism
Protein Ammonia Urea
Uric Acid is derived from purine( a component of DNA ) catabolism
Decreased Creatinine Clearance associated with decreased Glomerular Filtration
Don’t forget to divide V by 1440 !

26. Reference Ranges BUN 10 - 20 mg / dl Creatinine 0.5 - 1.5 mg /dl
Uric Acid mg / dl
Creatinine Clearance ml / min
Ammonia ug / dl
BUN / Creat Ratio to 1