NONPROTEIN NITROGEN COMPOUNDS

Renal function Tests

The urinary system includes kidneys, ureters, the bladder and the urethra. The kidneyis a vital organ that performs three important tasks: Excretory Homeostatic Endocrine function.

Three tests for renal function: 1. Assessment of Glomeruli Filtration Rate Clearnce Tests 2. Assessment of Renal Vlood Flow- NPN determination 3. Assessment of Tubular Function Tests-(1) measuring the concentration and dilution of urine, (2) assessment of renal concentrating ability, (3) assessment of renal diluting capacity, and (4) assessment of urinary acidication.

Nonprotein Nitrogen Compounds

Urea

Amino Acid

Creatinine

Uric Acid

Nonprotein Nitrogen Compounds

Compound Urea Amino Acids Uric Acid Creatinine Creatine Ammonia
Approximate Plasma Concentration (% of Total NPN) Approximate Urine Concentration (% of Excreted N)

45-50 25 10 5 1-2 0.2

86.0 --1.7 4.5 --2.8

UREA
1.

2.
3. 4. 5.

Physiology Clinical application Methods Specimen Requirement Pathophysiology

1. Physiology
Waste product of the protein catabolism Excreted by the kidneys

Reactions involved in Urea cycle: Step 1: Formation of carbamoyl phosphate (catalyzed by Carbamoyl Phosphate Synthetase), in liver mitochondria

Step 2: Formation of Citrulline (catalyzed by Ornithine TransCarbamoylase) in liver mitochondria

Step 3: Formation of Argininosuccinate (catalysed by ArgininosuccinateSynthetase) in liver cytoplasm.

Step 4: Cleavage of arginosuccinate to form Arginine (catalyzed by Argininosuccinase, or ArgininosuccinateLyase) in liver cytoplasm.

Step 5: Cleavage of arginine to release urea (catalyzed by Arginase) in liver cytoplasm

Overall equation of the urea cycle 2NH3 + CO2 + 3ATP + 3H2O urea + 2 ADP + 4Pi + AMP

Fate of urea Urea which is the waste product of the urea cycle diffuses from the liver and is transported in the blood. The kidney filtered the urea from blood to be excreted as a component of urine. Small portion of urea diffuses from blood into the intestine where it is converted to carbon dioxide (CO2) and ammonia (NH3) by bacterial urease. The ammonia is either excreted as a component of the feces or reabsorbed in the blood.

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
disappearance of absorption is measured at 340 nm

Principle
Urea + 2 H2O Urease 2 NH4+ + CO3 2 GLDH Glutamate + NAD+ + H2O NH4+ + pH Indicator color change a. Nesslers reaction

i. GLDH coupled enzymatic NH + + 2-oxoglutarate + NADH + H+ 4

ii. Indicator dye

Ammonia + Nesslers salt Gum ghatti yellow

a. Berthelot reaction
Ammonia + alkaline hypochlorite Na nitoprusside indophenol blue

iii. Conductimetric

Conversion of unionized urea to NH4+ and CO32- results in increased conductivity

3. Method of Analysis
Chemical Method
i. Fearons reaction

Principle

Urea + DAM (Diacetyl Monoxime Method) 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
Prerenal azotemia Renal azotemia
Caused by reduce blood flow Congestive heart failure, shock, hemorrhage, dehydration, protein catabolism, high-protein diet

Damage of filtering structures of the kidney

Renal failure and renal disease (glomerular nephritis, tubular necrosis)

Postrenal azotemia

Urinary tract obstruction

Renal calculi, tumors of the bladder or protate

5. Pathophysiology
Decreased Concentration
Low protein intake Severe vomiting and diarrhea Liver disease Pregnancy

URIC ACID
1.

2.
3. 4. 5. 6.

Physiology Synthesis Clinical application Methods Specimen requirements 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.

Degradation of purines to nitrogenous excretory products

Two-thirds of the uric acid produced daily is being excreted by the kidneys and the remaining one-third is excreted in the stool. Overproduction of uric acid and/or underexcretion by the kidneys leads to the excess storage in the joints, tissue and organs causing inflammatory response. This reaction results to a condition characterized by painful joint(s) known as a gout attack. Urate crystals may also appear as kidney stones and lead to painful obstruction of the urinary tract. Uric acid that is not excreted can lead to its catabolism to allantoin, allantoic acid, urea, or ammonia.
Catabolism of uric acid

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
Phosphotungstic acid (Caraway method)

Principle
Uric Acid + H3PW12O4o + O2 -Na2CO3/OH- allantoin + tungsten blue + CO2

3. Method
Enzymatic Methods
First step Spectrophotometric (Blauch and Koch)

Principle
Uric Acid + O2 +2 H2O Uricase allantoin + CO2 + H2O2 Decrease in absorbance at 293 nm is measured (Uric acid v. allantoin) Catalase catalyzed a chemical indicator reaction H2O2 + + reagent colored compund Peroxidase H2O2 + indicator dye colored compound

Coupled enzyme (I)

Coupled enzyme (II)

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

Adult

Urine/ 24 hour

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

1. Physiology of Creatinine
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 X GFR = PCrt A

3. Method of Analysis
Chemical Method Direct Jaffe Reaction Jaffe-kinetic Jaffe with adsorbent (Lloyds method)

Principle
Creatinine + picrate red-orange complex Detection of color formation timed to avoid interference of noncreatinine chromogens Creatine in protein-free filtrate adsorbed onto Fullers earth (aluminum magnesium silicate); then reacted with alkaline picrate

Jaffe without adsorbent

Creatine in protein-free filtrate reacts with alkaline picrate to form colored complex

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

3. Method of Analysis
Enzymatic Method

Principle

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 sarcosine + O2 + H2O ADP sarcosine oxidase glycine + CH2O + H2O2 H2O2+ colorless substrate Peroxidase Colored product + H2O

CreatininaseH2O2

3. Method of Analysis
Specimen Adult Male Adult Female Child Adult Male
Urine 24 hour
Plasma or serum

Jaffe Method
0.9 1.3 mg/dL (80-115 mol/L) 0.6 1.1mg/dL (53-97 mol/L)

Enzymatic Mtd.
0.6 1.1 mg/dL (55-96 mol/L) 0.5 0.8 mg/dL (40-66 mol/L)

0.3 0.7 mg /dL (27- 0.0 0.6 mg/dL 62 mol/L) (0-52 mol/L) 800 2,000 mg/day 600 1,800 mg/day

Adult Female

5. Pathophysiology
Increased Concentration Renal failure (glomerular function) Plasma Concentration GFR

1. Physiology of Ammonia
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 Reyes syndrome acute metabolic disorder of the liver Inherited deficiencies of urea cycle

3. Method
Chemical Method
Ion-selective electrode

Principle Diffusion of NH3 through selective membrane into NH4Cl causing pH change, which is measured potentiometrically

Spectrophotometric

NH3 + bromphenol blue blue dye

Principle NH4+ + 2-oxoglutarate + NADPH + H+ GLDH Glutamate + NADP+ + H2O

Enzymatic Method
GLDH Decrease in absorbance is measured at 340 nm

3. Method
Specimen Adult Child (10 days to 2 yrs)
Plasma

Reference Values
19-60 g/dL 11-35 mol/L

68-136 g/dL 40-80 mol/L

4. Specimen Requirements
Specimen Considerations
1. May be measured using heparinized and EDTA tubes 2. Samples should be centrifuged at 0C to 0C within 20 minutes of collection and the plasma or serum removed

3. Avoid cigarette smoking for several hours