NoNprotein

nitrogen
compounds
 Nonprotein Nitrogen Compounds

Approximate Plasma Approximate Urine

Compound Concentration Concentration
(% of Total NPN) (% of Excreted N)
Urea 45-50 86.0
Amino Acids 25 ---
Uric Acid 10 1.7
Creatinine 5 4.5
Creatine 1-2 ---
Ammonia 0.2 2.8
UREA

1. Physiology
2. Clinical application
3. Methods
4. Specimen Requirement
5. Pathophysiology
1. Physiology

Waste product of the protein catabolism

Excreted by the kidneys
 2. Clinical Application
Clinical Application
Evaluate renal function
Asses hydration status
Determine nitrogen balance
Diagnosis of renal disease
Verify adequacy of dialysis
Conversion
• Urea N (mg/dL) ↔ urea (mg/dL)
1 urea N  2.14 urea
0.467 urea  urea N
• 0.357 mg/dL  mmol/L
 3. Method of Analysis
Enzymatic Method
First step
i. GLDH coupled enzymatic NH + + 2-oxoglutarate + NADH + H+
4
disappearance of absorption
is measured at 340 nm
ii. Indicator dye
iii. Conductimetric
Principle
Urea + 2 H2O –Urease 2 NH4+ + CO3 2-
 GLDH  Glutamate + NAD+ + H2O
NH4+ + pH Indicator  color change
a. Nessler’s reaction
Ammonia + Nessler’s salt –Gum ghatti yellow
a. Berthelot reaction
Ammonia + alkaline hypochlorite
–Na nitoprusside indophenol blue
Conversion of unionized urea to NH4+ and
CO32- results in increased conductivity
 3. Method of Analysis

Chemical Method Principle

Urea + DAM (Diacetyl Monoxime Method)
i. Fearon’s reaction
 yellow solution (Diazine dirivative)
Comment: Non-specific, uses toxic regents
 4. Specimen Requirements

Specimen Considerations
1. Use fasting blood sample since a high protein diet affects urea
2. Avoid fluoride or citrate anticoagulants since they inhibit urease
3. Refrigerate samples to avoid bacterial decomposition
 5. Pathophysiology
 Azotemia – ↑ urea in the blood
 Uremia – ↑ plasma urea accompanied by renal failure

Increased Concentration
• Caused by reduce blood flow
Prerenal
• Congestive heart failure, shock, hemorrhage,
azotemia dehydration, ↑ protein catabolism, high-protein diet
• Damage of filtering structures of the kidney
Renal
• Renal failure and renal disease (glomerular nephritis,
azotemia
tubular necrosis)
Postrenal • Urinary tract obstruction
azotemia • Renal calculi, tumors of the bladder or protate
 5. Pathophysiology

Decreased Concentration
• Low protein intake
• Severe vomiting and diarrhea
• Liver disease
• Pregnancy
 Nonprotein Nitrogen Compounds

Approximate Plasma Approximate Urine

Compound Concentration Concentration
(% of Total NPN) (% of Excreted N)
Urea 45-50 86.0
Amino Acids 25 ---
Uric Acid 10 1.7
Creatinine 5 4.5
Creatine 1-2 ---
Ammonia 0.2 2.8
URIC ACID

1. Physiology
2. Clinical application
3. Methods
4. Specimen requirements
5. Pathophysiology
 1. Physiology
1

Major end-product of purine catabolism primarily in the liver.

After the blood is filtered at the glomerulus, the resulting fluid
enters the tubules of the kidneys to be secreted as urine.
 2. Clinical Application
1

Asses inherited disorders of purine metabolism

Confirm diagnosis and monitor treatment of gout
Diagnosis of renal calculi
Prevent uric acid nephropathy during chemotheraphy
Detect kidney disfunction
 3. Method

Chemical Method Principle

Uric Acid + H3PW12O4o + O2
Phosphotungstic acid
-Na2CO3/OH-  allantoin + tungsten
(Caraway method)
blue + CO2
 3. Method

Enzymatic Methods Principle

Uric Acid + O2 +2 H2O –Uricase
First step
allantoin + CO2 + H2O2
Spectrophotometric Decrease in absorbance at 293 nm
(Blauch and Koch) is measured (Uric acid v. allantoin)
Catalase – catalyzed a chemical
indicator reaction
Coupled enzyme (I)
H2O2 + + reagent  colored
compund
Peroxidase
Coupled enzyme (II) H2O2 + indicator dye  colored
compound
3. Method

Reference Intervals
(0.21-0.43
Adult Male 3.5 – 7.2 mg/dL
mmol/L)
Plasma or 0.16-0.36
Adult Female 2.6 – 6.0 mg/dL
serum mmol/L
0.12-0.33
Child 2.0-5.5 mg /dL
mmol/L
Urine/ 11.5-4.4
Adult 250-750, mg/day
24 hour mmol/day
 4. Specimen Requirements

Specimen Considerations
1. May be measured using heparinized plasma, serum or urine
2. Avoid gross lipemia, high bilirubin concentration and hemolysis
3. Avoid EDTA or flouride additives (affects uricase method)
 5. Pathophysiology

Increased Concentration (Hyperurecemia)

Enzyme deficiencies
Lesch-Nyhan syndrome
Phosphoribosylpyrophosphate synthetase deficiency
Glycogen storage disease type 1 (Glucose-6-phosphatase deficiency)
Fructose intolerance (fructose-1-phosphate aldolase deficiency)
Treatment of myeloproliferative disease w/ cytotoxic drugs
 5. Pathophysiology

Increased Concentration (Hyperurecemia)

Hemolytic and proliferative process
Chronic renal disease
Toxemia of pregnancy
Lactic acidosis
Drugs and poisons
Purine-rich diet
Increase tissue catabolism or starvation
 5. Pathophysiology

Decreased Concentration (Hypourecemia)

Liver disease
Defective tubular reabsorption (Fanconi sydrome)
Chemotheraphy with azathioprine or 6-mercaptopurine
Overtreatment with allopurinol
 Nonprotein Nitrogen Compounds

Approximate Plasma Approximate Urine

Compound Concentration Concentration
(% of Total NPN) (% of Excreted N)
Urea 45-50 86.0
Amino Acids 25 ---
Uric Acid 10 1.7
Creatinine 5 4.5
Creatine 1-2 ---
Ammonia 0.2 2.8
 1. Physiology
1

Chief product of muscle metabolism

Not affected by protein diet

Muscle Liver
 2. Clinical Application
1

Determine sufficiency of kidney function

Determine severity of kidney damage
Monitor the progression of kidney disease
Measure completeness of 24-hour urine
2. Clinical Application
Renal Clearance and Glomerular Filtration Rate
Glomerular filtration rate Volume of plasma filtered
(V) by the glomeruli per
unit of time

V
GFR =
t

UCrVu 1.73
GFR = X
PCrt A
 3. Method of Analysis

Chemical
Principle
Method
Direct Jaffe Reaction Creatinine + picrate  red-orange complex
Detection of color formation timed to avoid
Jaffe-kinetic
interference of noncreatinine chromogens
Creatine in protein-free filtrate adsorbed onto
Jaffe with adsorbent
Fuller’s earth (aluminum magnesium silicate);
(Lloyd’s method) then reacted with alkaline picrate
Jaffe without Creatine in protein-free filtrate reacts with
adsorbent alkaline picrate to form colored complex
 4. Specimen Requirements

Specimen Considerations Specimen Considerations

1. Glucose
2. α-ketoacids
3. Ascorbate
A. Flasely increase due to
4. Uric Acid
5. Cephalosporins
6. Dopamine
1. Bilirubin
B. Falsely decrease results due to 2. Hemoglobin
3. Lipemic specimens
 3. Method of Analysis

Enzymatic
Principle
Method
Creatinine + H2O —Creatininase Creatine
Creatine + ATP CK creatine phosphate + ADP
Creatininase-CK
Phosphoenolpyruvate + ADP —PK pyruvate + ATP
Pyruvate + NADH + H+ LD Lactate + NAD+
Creatinine + H2O —Creatininase Creatine
Creatine H2O —Creatininase Sarcosine + ADP
Creatininase- sarcosine + O2 + H2O ADP —sarcosine oxidase
H2O2 glycine + CH2O + H2O2
H2O2+ colorless substrate —Peroxidase  Colored
product + H2O
 3. Method of Analysis

Specimen Jaffe Method Enzymatic Mtd.

0.9 – 1.3 mg/dL 0.6 – 1.1 mg/dL
Adult Male
(80-115 µmol/L) (55-96 µmol/L)
Adult Plasma or 0.6 – 1.1mg/dL 0.5 – 0.8 mg/dL
Female serum (53-97 µmol/L) (40-66 µmol/L)
0.3 – 0.7 mg /dL (27- 0.0 – 0.6 mg/dL
Child
62 µmol/L) (0-52 µmol/L)
Adult Male 800 – 2,000 mg/day
Urine
Adult 24 hour 600 – 1,800 mg/day
Female
 5. Pathophysiology

Increased Concentration
Renal failure (glomerular function)
↑ Plasma Concentration  ↓ GFR
 Nonprotein Nitrogen Compounds

Approximate Plasma Approximate Urine

Compound Concentration Concentration
(% of Total NPN) (% of Excreted N)
Urea 45-50 86.0
Amino Acids 25 ---
Uric Acid 10 1.7
Creatinine 5 4.5
Creatine 1-2 ---
Ammonia 0.2 2.8
 1. Physiology
1

By product of amino acid

deamination.
Remove from the circulation and
converted to urea in the liver.
 2. Clinical Application
1

Diagnosis of hepatic failure

Reye’s syndrome – acute metabolic disorder of the liver
Inherited deficiencies of urea cycle
 3. Method

Chemical Method Principle

Diffusion of NH3 through selective
Ion-selective electrode membrane into NH4Cl causing pH change,
which is measured potentiometrically
Spectrophotometric NH3 + bromphenol blue  blue dye

Enzymatic Method Principle

GLDH
NH4+ + 2-oxoglutarate + NADPH + H+
Decrease in absorbance
GLDH Glutamate + NADP+ + H2O
is measured at 340 nm
3. Method

Specimen Reference Values

Adult 19-60 µg/dL 11-35 µmol/L
Child Plasma
68-136 µg/dL 40-80 µmol/L
(10 days to 2 yrs)
 4. Specimen Requirements

Specimen Considerations
1. May be measured using heparinized and EDTA tubes
2. Samples should be centrifuged at 0°C to 0°C within 20 minutes of
collection and the plasma or serum removed
3. Avoid cigarette smoking for several hours
 Nonprotein Nitrogen Compounds

Approximate Plasma Approximate Urine

Compound Concentration Concentration
(% of Total NPN) (% of Excreted N)
Urea 45-50 86.0
Amino Acids 25 ---
Uric Acid 10 1.7
Creatinine 5 4.5
Creatine 1-2 ---
Ammonia 0.2 2.8