Non protein nitrogen

Non protein nitrogen

1. 1
CLINICAL CHEMISTRY
NON - PROTEIN NITROGEN
Presented by: Shahid Nawaz
Researcher
King Saud University
Riyadh

2. 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. 3
Key Terms
•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. 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. 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. 6
•BUN ( Blood Urea Nitrogen )BUN ( Blood Urea Nitrogen )
–Blood Urea Nitrogen = BUNBUN = 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 CO
2
–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. 7
–BUN disease correlationsBUN disease correlations
•Azotemia = Elevated plasma BUN
•PrerenalPrerenal ­­ BUN 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
•PrerenalPrerenal ¯¯ BUN BUN ( Not related to renal function )
–Decreased dietary protein
–Increased protein synthesis ( Pregnant women , children )

8. 8
–RenalRenal causes of causes of ­­ BUN BUN
•Renal disease with decreased glomerular filtration
–Glomerular nephritis
–Renal failure form Diabetes Mellitus
–Post renalPost renal causes of causes of ­­ BUN ( not related to renal function ) BUN ( not related to renal function )
•Obstruction of urine flow
– Kidney stones
–Bladder or prostate tumors
–UTIs

9. 9
•BUN / Creatinine RatioBUN / Creatinine Ratio
–Normal BUN / Creatinine ratio is 10 – 20 to 1Normal BUN / Creatinine ratio is 10 – 20 to 1
–Creatinine is another NPNCreatinine is another NPN
–Pre-renal increased BUN / Creat ratio
–BUN is more susceptible to non-renal factors
–Post-renalPost-renal increased ratio BUN / Creat ratio
–Both BUN and Creat are elevated
–RenalRenal decreased BUN / Creat ratio
– Low dietary protein or severe liver disease
Increased BUN
Normal Creat
Increased BUN
Increased Creat
Decreased BUN
Normal Creat

10. 10
–BUN analytical methodsBUN analytical methods
•BUN is an old term, but still in common useBUN 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 )
UREA
2 NH
4
+
+ HCO
3
-
Urease
NH
4
+
+ 2-OXOGLUTARATE
GLDH
GLUTAMATE
NADH NAD
Measure the rate of decreased absorbance at 340 nm
NADH absorbs … NAD does not absorb
Reference range : 10 – 20 mg / dl

11. 11
•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. 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. 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 : 0.5 - 1.5 mg / dl

14. 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. 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. 16
–Uric acid analysis
Uric acid + O
2
+ H
2
O
Allantoin + CO
2
Uricase
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
+H
2
O
2
Reference range : 3.5 - 7.2 mg/dl (males)
2.6 - 6.0 mg/dl (females)
Let’s remember 3.0 - 7.0 mg/dl

17. 17
•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

18. 18
Ammonia analytical techniques
NH
4
+
+ 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

19. 19
•Creatinine Clearance
–Calculated measurement of the rate at which creatinine is removed from
the plasma by the kidneys
– Measurement of glomerular filtration ( renal function )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. 20
24 Hour Urine collection
Container.
The volume can be measured
directly off the container.

21. 21
–Creatinine Clearance specimens
•24 hour urine specimen
•Plasma / serum creatinine collected during the urine collection
•24 Hour Creatinine Clearance Formula
•CREATININE CLEARANCE = ÷
ø
ö
ç
è
æ
÷
ø
ö
ç
è
æ
A
U 73.1
P
V
U = Creatinine concentration of the 24 hour urine ( mg / dl )
V = 24 hour urine volume ( mls ) per minute - V / 1440 = mls / minute 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. 22
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
1.73 1.73 1440
5.0 2.05
UV
Creat Cl
P A
æ ö æ ö
= = =
ç ¸ ç ¸
è ø è ø
Creat Cl = 2 ml / min …. Very poor clearance !!!

23. 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. 24
–Reference range
•97 - 137 ml / min ( male)
•88 - 128 ml / min (female)
•Let’s remember 90 - 130 ml / min

25. 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
÷
ø
ö
ç
è
æ
÷
ø
ö
ç
è
æ
=
AP
UV 73.1
Clearance Creatinine
Don’t forget to divide V by 1440 !

26. 26
Reference Ranges
•BUN 10 - 20 mg / dl
•Creatinine 0.5 - 1.5 mg /dl
•Uric Acid 3.0 - 7.0 mg / dl
•Creatinine Clearance90 - 130 ml / min
•Ammonia 20 - 60ug / dl
•BUN / Creat Ratio 10 - 20 to 1