NONPROTEIN

NITROGEN
COMPOUNDS
KIDNEY

• Paired bean shaped organs

• Nephrons
• Elimination of Waste Products
• Maintenance of Blood Volume
• Maintenance of Electrolyte
Balance
• Maintenance of Acid base balance
• Endocrine Function
Nitrogen Containing Compounds

Urea Amino Acid

Creatinine Uric Acid

 Nonprotein Nitrogen Compounds

Plasma Conc. Urine Conc.

Compound
(% 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 of Analysis
4. Specimen Requirement
5. Pathophysiology
1. Physiology

Major waste product of the protein catabolism

Excreted by the kidneys
2. Clinical Application
Clinical Application Conversion
Renal function • Urea N (mg/dL) ↔ urea (mg/dL)
Hydration status
• 1 urea N  2.14 urea
Nitrogen balance
• 0.467 urea  1 urea N
Renal disease
Adequacy of dialysis • 0.357 mg/dL  mmol/L
3. Method of Analysis

Enzymatic Method Chemical Method

i. GLDH coupled enzyme i. Fearon’s reaction
ii. Indicator dye
iii. Conductimetric (ISE)
 3. Method of Analysis
Enzymatic Method Principle
First step Urea + 2 H2O –Urease 2 NH4+ + CO3 2-
i. GLDH coupled enzyme
NH4+ + 2-oxoglutarate + NADH + H+
disappearance of
 GLDH  glutamate + NAD+ + H2O
NADH at 340 nm
NH4+ + pH Indicator  color change
ii. Indicator dye a. Nessler’s reaction (HgI2/KI)
b. Berthelot’s
Berthelot reaction
reaction(Alk.
(Alk.hypochlorite
hypochlorite) )
Conversion of unionized urea to NH4+ and
iii. Conductimetric (ISE)
CO32- results in increased conductivity
 3. Method of Analysis
Chemical Method Principle
Urea + DAM (Diacetyl Monoxime Mtd.) 
i. Fearon’s reaction
Yellow solution (Diazine dirivative)
3. Method of Analysis

Enzymatic Method Chemical Method

i. GLDH coupled enzyme i. Fearon’s reaction
ii. Indicator dye
iii. Conductimetric (ISE)
3. Method of Analysis

Reference Intervals
Plasma or
Adult 6 – 20 mg/dL 2.1-7.1 mmol/L
serum
Urine, 11.5-4.4
12-20 g/day
24 hour mmol urea/day
4. Specimen Requirements

Specimen Considerations
1. Use fasting blood
2. Avoid fluoride or citrate anticoagulants
3. Refrigerate Samples
5. Pathophysiology

Azotemia (↑ urea in the blood)

Prerenal • Reduce blood flow or ↑ protein catabolism
Renal (Uremia) • Damage of nephrons (Renal failure/disease)
Postrenal • Urinary tract obstruction (calculi, tumors)
 5. Pathophysiology

Decreased Concentration
• Low protein intake
• Severe vomiting and diarrhea
• Liver disease
• Pregnancy
UREA
1. Physiology
2. Clinical application
3. Methods of Analysis
4. Specimen Requirement
5. Pathophysiology
 Nonprotein Nitrogen Compounds

Plasma Concentration Urine Concentration

Compound (% 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
1

Major end-product of purine catabolism.

Present as monosodium urates in plasma (98-100% reabsorbed)
May precipitate in tissues (blood pH ≈ 7 ; BUA > 6.8 mg/dL )
 2. Clinical Application
11

• Inherited disorders of purine metabolism

• Gout
• Renal calculi
• Uric acid nephropathy (chemotheraphy)
• Kidney dysfunction

Uric acid crystals in urine

3. Method

Enzymatic Methods Chemical Method

Spectrophotometric Phosphotungstic acid
(Blauch and Koch) (Caraway method)
Coupled enzyme (I)
Catalase
Coupled enzyme (II)
Peroxidase
 3. Method

Enzymatic Methods Principle

Uric Acid + O2 +2 H2O –Uricase
First step
allantoin + CO2 + H2O2
Spectrophotometric ↓ absorbance at 293 nm
(Blauch and Koch) (Uric acid v. allantoin)
Coupled enzyme (I) H2O2 + reagent –Catalase colored
Catalase compound
Coupled enzyme (II) H2O2 + indicator dye –Peroxidase
Peroxidase Red dye
 3. Method

Chemical Method Principle

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

Enzymatic Methods Chemical Method

Spectrophotometric Phosphotungstic acid
(Blauch and Koch) (Caraway method)
Coupled enzyme (I)
Catalase
Coupled enzyme (II)
Peroxidase
3. Method

Reference Intervals
mg/dL mmol/L
Adult Male 3.5 – 7.2 0.21 – 0.43
Plasma
Adult Female 2.6 – 6.0 0.16 – 0.36
or serum
Child 2.0 – 5.5 0.12 – 0.33
Reference Intervals

Urine / 1.5 – 4.4

Adult 250-750 mg/day
24 hour mmol/day
4. Specimen Requirements

Specimen Considerations
1. Heparinized plasma, serum or urine
2. Lipemia, bilirubin, hemolysis (↓ UA)
3. Salicylates and thiazides (↑ UA)
 5. Pathophysiology

Hyperuricemia (Enzyme deficiencies)

1. Lesch-Nyhan syndrome
• ↓ HGPRT
• ↓ reutilization of purine, ↑ synthesis of purine
2. Glycogen storage disease type 1
• ↓ G-6-P
• ↑ triglycerides, ↓ renal urate excretion
3. Fructose intolerance
• ↓ F1PA
• ↑ lactate, ↓ renal urate excretion
 5. Pathophysiology

Hyperuricemia
4. Hemolytic and proliferative process
• ↑ metabolism of cell nuclei
5. Treatment of myeloproliferative disease w/ cytotoxic drugs
• ↑ destruction of cell
6. Chronic renal disease
• ↓ uric acid filtration and secretion
7. Toxemia of pregnancy and lactic acidosis
• ↑ binding to renal tubules
8. Purine-rich diet
 5. Pathophysiology

Hypouricemia
1. ↓ PRPP Synthetase
• ↓ de novo purine synthesis
2. Liver disease
• ↓ uric acid synthesis
3. Fanconi sydrome
• Defective tubular reabsorption
4. Chemotheraphy with azathioprine or 6-mercaptopurine
• ↓ de novo purine synthesis
5. Overtreatment with allopurinol
• ↓ XO, ↓ de novo purine synthesis
 Nonprotein Nitrogen Compounds

Plasma Concentration Urine Concentration

Compound (% 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
Creatinine
1. Physiology
2. Clinical application
3. Methods
4. Specimen requirements
5. Pathophysiology
 1. Physiology
1

Chief product of muscle metabolism

Not affected by protein diet

Muscle Liver
2. Clinical Application
1
1v
1

1. Sufficiency of kidney function

2. Severity of kidney damage
3. Progression of kidney disease
4. Completeness of 24-hour urine
2. Clinical Application
Renal Clearance and Glomerular Filtration Rate
Volume of plasma filtered
Glomerular filtration rate
(V) by the glomeruli per
(Creatinine clearance)
unit of time (mL/minute)

V
GFR =
t

UCrVu 1.73
GFR = X
PCrt A
2. Clinical Application
Calculate the creatinine clearance
Serum creatinine = 1.2 mg/dL Urine creatinine = 120 mg/dL
Urine volume = 1.75 L/day Surface Area = 1.80 m2

UCrVu 1.73
GFR = X
PCr t A
UCr 1.75 L 1 Day 1000mL 1.73 m2
GFR = X X X
PCr Day 1440min L 1.80 m2
120mg/dL 1750mL 1.73m2
GFR = X
1.2 mg/dL 1440min 1.80m2
 3. Method of Analysis

Chemical Method Principle

Creatinine + picrate 
1. Jaffe Reaction
Red-orange complex
Rate of change of absorbance
2. Jaffe-kinetic
(color formation)
PFF is adsorbed first onto Fuller’s
3. Jaffe with adsorbent
earth (aluminum magnesium silicate)
 3. Method of Analysis

Specimen Jaffe Method

Unit mg/dL µmol/L
Adult Male 0.9 – 1.3 80-115
Plasma or
Adult Female 0.6 – 1.1 53-97
serum
Child 0.3 – 0.7 27-62
Adult Male 800 – 2,000 mg/day
Urine 24 hour
Adult Female 600 – 1,800 mg/day

BUN / Creatinine Ratio : 10:1 to 20:1

4. Specimen Requirements

Specimen Considerations
1. Glucose
2. α-ketoacids
3. Ascorbate
A. Falsely ↑
4. Uric Acid
5. Cephalosporins
6. Dopamine
1. Bilirubin
B. Falsely ↓ 2. Hemoglobin
3. Lipemic specimens
5. Pathophysiology

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

Plasma Concentration Urine Concentration

Compound (% 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
11

By product of amino acid deamination.

Converted to urea in the liver.
2. Clinical Application
1

1. Hepatic failure and hepatic coma

2. Reye’s syndrome
3. Inherited deficiencies of urea cycle
 3. Method

Chemical Method Principle

Diffusion of NH3 through
Ion-selective electrode selective membrane into
NH4Cl causing pH change.
Spectrophotometric NH3 + bromphenol blue  blue dye

Enzymatic Method Principle

GLDH NH4+ + 2-oxoglutarate + NADPH + H+
↓ absorbance (340 nm) GLDH Glutamate + NADP+ + H2O
 3. Method
Specimen Reference Values
µg/dL µmol/L
Adult 19-60 11-35
Plasma
Child (10 days - 2 yrs) 68-136 40-80
 4. Specimen Requirements

Specimen Considerations
1. Heparinized and EDTA tubes
2. Samples is centrifuged at 0-4°C within 20 mins.
of collection and the plasma/serum removed
3. Avoid cigarette smoking for several hours
 Nonprotein Nitrogen Compounds

Plasma Concentration Urine Concentration

Compound (% 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
 DISEASE CORRELATION

AZOTEMIA
Elevated nitrogenous compound
pre renal azotemia
-diminished GFR with normal renal function
-  BUN  renal blood flow  GFR  tubular reabsorption = slower
filtrate flow
Congested heart Failure, Dehydration Shock
renal azotemia
- Damaged within the kidneys
-  GFR BUN , slowly rising creatinine value
post renal azotemia
- Usually result of urinary tract obstruction
-  GFR,  CREA  BUN back diffusion of urea into the circulation
 DISEASE CORRELATION

UREMIA
Clinical syndrome comprised of a marked elevation in plasma urea
and other nitrogenous waste products, accompanied by acidemia
and electrolyte imbalance
Kidneys fail to eliminate waste products of metabolism
 BUN, CREA,  K
BUN: CREA RATIO

LOW < 10:1

Low protein Diet, Aute Tubular Necrosis, Repeated Dialysis, Hepatic Disease
HIGH > 20:1 , with normal Creatinine
Prerenal azotemia, Dehydration, Catabolic states, GI hemorrhage, High Protein Diet
HIGH > 20:1 with increased Creatinine
Postrenal azotemia, pre renal azotemia with renal disease, renal failure
 KIDNEY FUNCTION TEST

TEST FOR GLOMERULAR FILTRATION RATE

• Glomerular Filtration Rate

• Clearances
Inulin Clearance
Creatinine Clearance
Urea Clearance
• Phenolsulfonthalein Dye Test (PSP Dye Test)
• Cystatin C
TEST FOR RENAL BLOOD FLOW

 Blood Urea Nitrogen

 Creatinine
 Blood Uric Acid
TEST FOR MEASURING TUBULAR FUNCTION

Excretion Test
• Para Amino Hippurate Test (Diodrast Test)
-reference method for tabular function
• PSP Dye Test
-measures excretion of dye proportional to renal tubular mass

Concentration Test
reflects the function of the collecting tubules and the loops of Henle
• Specific Gravity
• Osmolality
expression of concentration in terms of the total number of solute particles
present/kg of solvent
direct method: freezing point osmometry, vapor pressure osmometry
indirect method (1.86 x Na + Glucose(mg/dl)/18 +BUN (mg/dl)/2.8