NPN (1)

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 Tests2. Assessment of Renal Vlood Flow- NPN determination3. 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 acidification.

Nonprotein Nitrogen Compounds

Urea

Uric AcidCreatinine

Amino Acid

Nonprotein Nitrogen Compounds

CompoundApproximate Plasma Concentration

(% of Total NPN)

Approximate Urine Concentration (% of Excreted N)

Urea 45-50 86.0Amino Acids 25 ---Uric Acid 10 1.7Creatinine 5 4.5Creatine 1-2 ---Ammonia 0.2 2.8

UREA

1. Physiology2. Clinical application3. Methods4. Specimen Requirement5. Pathophysiology

1. Physiology

Waste product of the protein catabolismExcreted 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 Trans-Carbamoylase) 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 cycle2NH3 + 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 ConversionEvaluate renal function • Urea N (mg/dL) ↔ urea (mg/dL)

1 urea N 2.14 urea

0.467 urea urea N• 0.357 mg/dL mmol/L

Asses hydration statusDetermine nitrogen balanceDiagnosis of renal diseaseVerify adequacy of dialysis

3. Method of Analysis

Enzymatic Method PrincipleFirst step Urea + 2 H2O –Urease 2 NH4

+ + CO3 2-

i. GLDH coupled enzymatic disappearance of absorption is measured at 340 nm

NH4+ + 2-oxoglutarate + NADH + H+

GLDH Glutamate + NAD+ + H2O

ii. Indicator dye

NH4+ + pH Indicator color change

a. Nessler’s reaction Ammonia + Nessler’s salt –Gum ghatti yellowb. Berthelot reaction Ammonia + alkaline hypochlorite

–Na nitoprusside indophenol blue

iii. ConductimetricConversion of unionized urea to NH4

+ and

CO32- results in increased conductivity

3. Method of Analysis

Chemical Method Principle

i. Fearon’s reaction Urea + DAM (Diacetyl Monoxime Method) yellow solution (Diazine dirivative)

Comment: Non-specific, uses toxic regents

4. Specimen Requirements

Specimen Considerations1. Use fasting blood sample since a high protein diet affects urea2. Avoid fluoride or citrate anticoagulants since they inhibit urease3. Refrigerate samples to avoid bacterial decomposition

5. Pathophysiology

Increased Concentration

Prerenal azotemia

• Caused by reduce blood flow• Congestive heart failure, shock, hemorrhage, dehydration, ↑ protein catabolism, high-protein diet

Renal azotemia

• 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

Azotemia – ↑ urea in the blood Uremia – ↑ plasma urea accompanied by renal failure

5. Pathophysiology

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

URIC ACID

1. Physiology2. Synthesis3. Clinical application4. Methods5. Specimen requirements6. Pathophysiology

1. Physiology1

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

Catabolism of uric acid

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 under-excretion 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.

2. Clinical Application1

Asses inherited disorders of purine metabolismConfirm diagnosis and monitor treatment of goutDiagnosis of renal calculiPrevent uric acid nephropathy during chemotheraphyDetect kidney disfunction

3. Method

Chemical Method Principle

Phosphotungstic acid(Caraway method)

Uric Acid + H3PW12O4o + O2

-Na2CO3/OH- allantoin + tungsten blue + CO2

3. Method

Enzymatic Methods Principle

First stepUric Acid + O2 +2 H2O –Uricase allantoin + CO2 + H2O2

Spectrophotometric(Blauch and Koch)

Decrease in absorbance at 293 nm is measured (Uric acid v. allantoin)

Coupled enzyme (I)Catalase – catalyzed a chemical indicator reactionH2O2 + + reagent colored compund

Coupled enzyme (II)PeroxidaseH2O2 + indicator dye colored compound

3. Method

Reference Intervals

Adult Male

Plasma or serum

3.5 – 7.2 mg/dL (0.21-0.43 mmol/L)

Adult Female 2.6 – 6.0 mg/dL 0.16-0.36 mmol/L

Child 2.0-5.5 mg /dL 0.12-0.33 mmol/L

Adult Urine/ 24 hour 250-750, mg/day 11.5-4.4

mmol/day

4. Specimen Requirements

Specimen Considerations1. May be measured using heparinized plasma, serum or urine2. 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 Creatinine1

Chief product of muscle metabolismNot affected by protein diet

LiverMuscle

2. Clinical Application1

Determine sufficiency of kidney functionDetermine severity of kidney damage

Monitor the progression of kidney disease

Measure completeness of 24-hour urine

2. Clinical Application

Renal Clearance and Glomerular Filtration RateGlomerular filtration rate Volume of plasma filtered

(V) by the glomeruli per unit of time

GFR =UCrVu

PCrt1.73A

X

GFR =Vt

3. Method of Analysis

Chemical Method Principle

Direct Jaffe Reaction Creatinine + picrate red-orange complex

Jaffe-kinetic Detection of color formation timed to avoid interference of noncreatinine chromogens

Jaffe with adsorbent (Lloyd’s method)

Creatine in protein-free filtrate adsorbed onto Fuller’s 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

A. Flasely increase due to

1. Glucose2. α-ketoacids3. Ascorbate4. Uric Acid5. Cephalosporins6. Dopamine

B. Falsely decrease results due to1. Bilirubin2. Hemoglobin3. Lipemic specimens

3. Method of Analysis

Enzymatic Method Principle

Creatininase-CK

Creatinine + H2O —Creatininase Creatine Creatine + ATP CK creatine phosphate + ADPPhosphoenolpyruvate + ADP —PK pyruvate + ATPPyruvate + NADH + H+ LD Lactate + NAD+

Creatininase-H2O2

Creatinine + H2O —Creatininase Creatine Creatine H2O —Creatininase Sarcosine + ADPsarcosine + O2 + H2O ADP —sarcosine oxidase glycine + CH2O + H2O2

H2O2+ colorless substrate —Peroxidase Colored product + H2O

3. Method of Analysis

Specimen Jaffe Method Enzymatic Mtd.

Adult Male

Plasma or serum

0.9 – 1.3 mg/dL (80-115 µmol/L)

0.6 – 1.1 mg/dL (55-96 µmol/L)

Adult Female

0.6 – 1.1mg/dL (53-97 µmol/L)

0.5 – 0.8 mg/dL (40-66 µmol/L)

Child 0.3 – 0.7 mg /dL (27-62 µmol/L)

0.0 – 0.6 mg/dL (0-52 µmol/L)

Adult MaleUrine

24 hour

800 – 2,000 mg/dayAdult Female 600 – 1,800 mg/day

5. Pathophysiology

Increased ConcentrationRenal failure (glomerular function)↑ Plasma Concentration ↓ GFR

1. Physiology of Ammonia1

By product of amino acid deamination.Remove from the circulation and converted to urea in the liver.

2. Clinical Application1

Diagnosis of hepatic failureReye’s syndrome – acute metabolic disorder of the liverInherited deficiencies of urea cycle

3. Method

Chemical Method Principle

Ion-selective electrodeDiffusion of NH3 through selective membrane into NH4Cl causing pH change, which is measured potentiometrically

Spectrophotometric NH3 + bromphenol blue blue dye

Enzymatic Method PrincipleGLDH Decrease in absorbance is measured at 340 nm

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

3. Method

Specimen Reference ValuesAdult

Plasma19-60 µg/dL 11-35 µmol/L

Child (10 days to 2 yrs) 68-136 µg/dL 40-80 µmol/L

4. Specimen Requirements

Specimen Considerations1. May be measured using heparinized and EDTA tubes2. Samples should be centrifuged at 0°C to 0°C within 20 minutes of

collection and the plasma or serum removed3. Avoid cigarette smoking for several hours

Thank You!