2. 2
Nonprotein Nitrogen
- The determination of nonprotein nitrogenous
substances in the blood has traditionally been
used to monitor renal function.
- The term nonprotein nitrogen (NPN) originated in
the early days of clinical chemistry when
analytical methodology required that protein be
removed from the sample before analysis.
3. 3
Nonprotein Nitrogen
- The NPN fraction is made up of about 15
compounds of clinical interest, the majority of
these compounds rise from the catabolism of
proteins and nucleic acids.
4. 4
Components of the Nonprotein-
Nitrogen Fraction
Approximate Plasma Concentration
Compound (% of Total NPN)
Urea 45
Aminoacids 20
Uric Acid 20
Creatinine 5
Creatine 1-2
Ammonia 0.2
5. 5
UREA
- Urea constitutes nearly half the nonprotein
nitrogen substances in the blood.
- It is synthesized in the liver from Co2 and the
ammonia arising from the deamination of amino
acids by means of the ornithine.
7. 7
UREA
- Urea constitutes the major excretory product of
protein metabolism, following synthesis in the
liver, urea is transported by the plasma to
kidney, where it is readily filtered from the
plasma by the glomerulus.
- Most of the urea in the glomerular filtrate is
excreted in the urine, and up to 40-50 % is
reabsorbed by passive diffusion during passage
of the filtrate through the renal tubules.
8. 8
UREA
- An elevated level of urea in the blood is called
azotemia.
- Very high levels of plasma urea accompanied by
renal failure is called uremia or the urermic
syndrome this is eventually fatal if not treated by
dialysis.
- The major causes of decreased plasma urea
levels include decreased protein intake and
severe liver disease.
9. 9
Analytical Methods
- Because measurements of urea were initially
done on a protein-free filtrate of whole blood,
and early analytical methods were based on
measuring the amount of nitrogen, this assay is
commonly called a BUN or blood urea nitrogen
determination.
10. 10
Analytical Methods
Two analytical approaches have been used to
assay for urea:
The oldest and most often used involves the
hydrolysis of urea by the enzyme urease and
quantitation of the NH+
4.
11. 11
Analytical Methods
Early colorimetric methods were based on using
Nesslers reagent or the Berthelot reaction to
detect the NH+
4 produced.
13. 13
Specimen Requirements
and Interfering Substances
- Fluoride or citrate will inhibit the urease utilized
in the coupled enzymatic methods.
- When collecting plasma, NH+
4 ions and high
concentrations of sodium fluoride or sodium
çitrate must be avoided.
- Urea concentration may be readily measured in
serum, plasma, or urine.
14. 14
Specimen Requirements
and Interfering Substances
- Fasting sample is usually not required.
- A non-hemolyzed sample is recommended.
- Urea is quite susceptible to bacterial
decomposition, so samples (especially urine)
that cannot be analyzed within a few hours
should be refrigerated.
16. 16
CREATININE
- Creatine is synthesized mainly in the liver from
arginine, glycine, and methionine.
- It is then transported to other tissues, such as
muscle, where it is converted to:
phosphocreatine, which serves as a high energy
source.
17. 17
CREATININE
- Either creatine phosphate or creatine, under
physiologic conditions, spontaneously loses
phosphoric acid or water, respectively, to form its
anhydride, creatinine which excreted into the
plasma.
19. 19
Analytical Methods for Creatinine
- The methods most frequendy used to measure
creatinine are based on the Jaffe reaction first
described in 1886, in this reaction, creatinine
reacts with picric acid in alkaline solution to form
a red-orange chromogen.
20. 20
Specimen Requirements and
Interfering Substances
- Creatinine can be measured in serum, plasma,
or urine.
- Hemolysis should be avoided, since there are a
significant number of nonspecific chromogens in
the red cells.
21. 21
Specimen Requirements and
Interfering Substances
- Fasting is not require.
- A heavy protein ingestion may elevate levels of
creatinine.
- Urine should be refrigerated after collection or
frozen if storage longer than four days is
required.
22. 22
Reference Interval
- Reference intervals vary with age and sex and may
decrease in the elderly.
Cord blood 0.6-1.2 mg/dl (53-106 mmol/L)
Children 0.3-0.7 mg/dl (27-62 mmol/L)
Adult
Male 0.6-1.2 mg/dl (53-106 mmol/L)
Female 0.5-1.1 mg/dl (44-97 mmol/L)
