1. Non-Protein Nitrogen (NPN) Compounds (Urea, Creatinine & Uric Acid)
Lab. 7

2. Introduction
Nitrogen containing compounds that are not proteins or polypeptides
Total NPN can be tested by making a protein-free filtrate
Useful clinical information is obtained from individual components of NPN fraction

3. Urea Highest concentration of NPN in blood (45%)
Major excretory product of protein metabolism
Urea is synthesized in the liver, from CO2 and ammonia, as the final product of amino acid catabolism.
It is freely filtered at the glomerulus, though 40% is passively reabsorbed by the proximal tubules.

4. Urea Blood urea levels can vary proportionately with:
the protein content of the diet,
rate of protein catabolism during tissue breakdown,
and liver function.
Because of its metabolism, urea is a nonspecific indicator of renal function.
However, in healthy persons with an average diet, the blood urea level is a very sensitive indicator of renal function.
Blood urea levels are usually elevated before significant changes in creatinine levels have occurred.
Monitoring blood urea levels is very useful when one is following the course of renal disease.

5. Clinical Significance
States associated with elevated levels of urea in blood are referred to as uremia or azotemia.
Causes of urea plasma elevations:
Prerenal
Renal
and postrenal
Parallel determination of urea and creatinine is performed to differentiate between pre-renal and post-renal azotemia.

6. Specimen
Serum and heparinized plasma can be used for the urease/GLDH methods.
Fluoride will inhibit the urease reaction; therefore methods employing urease cannot use serum preserved with fluoride.
Ammonium heparin also cannot be used as an anticoagulant for urease methods.
Stability in serum or plasma:
7 days at 4–8°C
1 year at -20°C
Because of urea’s susceptibility to bacterial degradation, serum and urine samples should be kept at 4° to 8° C until analysis.

7. Specimen
One can analyze urine after a 1:20 to 1:50 or 1:100 sample dilution, depending on the method and instrument employed.
One can also preserve urine samples by maintaining the pH at less than 4.
Stability in urine:
7 days at 4–8°C
1 month at -20°C
Discard contaminated specimens.

8. Urease/GLDH Method
The method is optimized for 2-point kinetic measurement.
Decrease in absorbance at 340 nm is proportional to concentration of urea

9. Creatinine
Creatinine is a non-protein nitrogen waste product formed in muscle.
Creatine Phosphate – phosphoric acid = Creatinine
Creatine – water = Creatinine
Filtered by kidney and excreted in the urine
Creatinine filters easily into the glomerular filtrate and is not reabsorbed by the tubule.
The amount excreted daily is a function of muscle mass and is not affected too much by diet

10. Clinical Significance
Elevated Creatinine is found with abnormal renal function (i.e. GFR)
Therefore, any condition that reduces the glomerular filtration rate will result in:
a lessened excretion from the body,
with a consequent rise in the concentration of creatinine in the blood.
The serum creatinine is a better indicator of renal function than either that of BUN or uric acid

11. Clinical Significance
For renal transplant patients, an increase in serum creatinine of 2 mg/L has been used as a criterion of establishing rejection.
In other persons a change in creatinine of 2 mg/L would represent a 20% loss in renal function.

12. Specimen One can analyze serum, plasma, or diluted urine.
The common anticoagulants (fluoride and heparin) do not cause interference, though heparin, which can be formulated as the ammonium salt, must be avoided in enzymatic methods that measure ammonia production.
Storage
7 days at 4-25oC
At least 3 months at -20oC

13. Specimen Urine should be diluted 1:100
Bacterial contamination has been found to falsely lower creatinine values measured using the Jaffé reaction.
The mechanism of this interference appears to be bacterial production of a substance that retards the rate of the Jaffé reaction.

14. Jaffe Method
Creatinine + Sodium Picrate Creatininr-picrate complex
( Yellow-orange)
The difference in absorbance at fixed times during conversion is proportional to the concentration of creatinine in the sample

15. Creatinine Clearance
Creatinine clearance is used to estimate the glomerular filtration rate (GFR).
Creatinine is chosen because it is freely filtered at the glomerulus and is not reabsorbed by the tubules.
However, a small amount of the creatinine (about 5%) in the final urine of healthy persons is derived from tubular secretion.
To do the test, one needs a precisely timed urine collection and a blood sample taken during the collection period.

16. Creatinine Clearance
Best results are obtained from a 24-h urine collection.
The test is initiated by having patients empty their bladder at the beginning of the timed period.
Urine is collected throughout the period, the bladder is again emptied at the end of the time period.

17. Creatinine clearance (mL/min)= (U x V)/P x 1.73/S
Creatinine determinations are performed on both samples. The creatinine clearance is calculated from the following formula:
Creatinine clearance (mL/min)= (U x V)/P x 1.73/S
Where:
U is urinary creatinine (mg/L),
V is volume of urine (mL/min),
P is plasma creatinine (mg/L),
S is the calculated surface area of the patient,
and is the surface area (m2) of a standard 70 kg person.

18. Creatinine Clearance
The range of creatinine clearance in healthy persons corrected to a surface area of m2 is 90 to 120 mL/min.
At low filtration rates, the creatinine clearance does not parallel true glomerular filtration rate because a relatively large portion of the urine creatinine is secreted rather than filtered.

19. Parallel determination of urea and creatinine is performed to differentiate between pre-renal and post-renal azotemia.
Pre-renal azotemia, caused by e.g. dehydration, increased protein catabolism or decreased renal perfusion, leads to increased urea levels, while creatinine values remain within the reference range.
In post-renal azotemias, caused by the obstruction of the urinary tract, both urea and creatinine levels rise, but creatinine in a smaller extent.

20. Uric Acid
Uric acid is formed from the breakdown of nucleic acids and is an end product of purine metabolism.
Uric acid is transported by the plasma from the liver to the kidney, where it is filtered and where about 70% is excreted.
The remainder of uric acid is excreted into the GI tract.

21. Clinical Significance
Disease states with increased plasma uric acid
Gout
Increased catabolism of nucleic acids
And renal disease
In Gout increased serum levels of uric acid lead to formation of monosodium urate crystals around the joints.
Uric acid test is useful to assess for gout and to monitor patients with renal failure

22. Clinical Significance
To monitor if uric acid levels are too high after chemotherapy or radiation.
Hypouricemia is seldom observed and associated with rare hereditary metabolic disorders.

23. Specimen Serum or plasma may be used Stability in serum / plasma:
6 months at -20°C
7 days at 4-8°C
3 days at 20-25°C

24. Enzymatic Colorimetric
Aminophenazone + DHBS
Uric acid is oxidized to allantoin by uricase.
The generated hydrogen peroxide reacts with 4-aminophenazone/ESPT to quinoneimine.