MLAB 2401: Clinical Chemistry

Renal Assessment

Nonprotein Nitrogen Compounds

• What are they?– Products from the catabolism of proteins and

nucleic acids– Consist of a molecule that contains nitrogen but

are not part of a protein– Useful to evaluate renal function

Clinically Significant NPN’sAnalyte Plasma Concentration (%)

Blood Urea Nitrogen (BUN) 45

Amino Acids 20

Uric Acid 20

Creatinine 5

Creatine 1-5

Ammonia 0.2

BUN

• Blood Urea Nitrogen – Urea is the nitrogenous end-product of protein &

AA metabolism.– Urea is formed in the liver when ammonia (NH3) is

removed and combined with CO2.– Rises quickly as compared to creatinine– Majority excreted in urine– Most widely used screening test of kidney

function

BUN: Clinical Significance

• Reference range 7-18 mg/dL• Decreased BUN

– Late pregnancy– Decreased protein intake– Severe liver disease– Overhydration

• Increased BUN– Azotemia

• Occurs when BUN concentration exceeds 20 mg/dL• Not always due to kidney malfunction

BUN / Creatinine Ratio– Normal Normal

BUN / Creatinine ratio is 12 – 20 to 1

– Pre-renal Azotemia Increased BUN due to non-renal causes

Congestive heart failure, high protein diets, dehydration Increased Ratio- BUN is high/ creatinine is normal

– RenalRenal Azotemia Disease directly affects nephron

Glomerulonephritis, Nephrotic syndrome, uremia, etc. Normal Ratio- both BUN and creatinine are proportionally elevated

– Post-renalPost-renal Azotemia Occurs after urine has left the kidney- due to obstruction Increased Ratio- BUN is high Plasma creatinine also elevated

–

Specimen Requirements: BUN

• Plasma• Serum• 24-hour Urine• nonhemolyzed

BUN: Methodology• Kjeldahl – a classical method for determining urea

concentration by measuring the amount of nitrogen present

• Berthelot reaction - Good manual method - that measures ammonia– Uses an enzyme (urease ) to split off the ammonia

• Diacetyl monoxide ( or monoxime)– Popular method but not well suited for manual

methods• because ➵ Uses strong acids and oxidizing

chemicals

Creatinine/Creatine

• Creatinine is formed from creatine and creatine phosphate in muscle

• Metabolic product cleared entirely by glomerular filtration

• Not reabsorbed• In order to see increased creatinine in serum,

50% kidney function is lost• Creatinine levels are affected by muscle mass,

creatine turnover, and renal function

Advantages of Creatinine

• Formed at a constant rate• Readily excreted• Not reabsorbed• Not affected by diet

Reference Range/Significance: Creatinine

Significance

• Evaluates renal function• Follows progress of renal

disease

• Increased results – Renal disease– Decrease in GFR– Obstruction in urinary system– Decreased muscle mass

Reference Range

• Urine– 0.8-2.0gm/ 24 hour

• Serum– 0.5-1.5mg/dL

Specimen requirements: Creatinine

• Plasma• Serum• Urine ( 24 hour or random)• Avoid hemolysis• Avoid icterus

Creatinine: Methodology• Jaffe reaction

– basic reaction for creatinine– Kinetic

• Principle: Protein-free filtrate(serum/urine) mixed with alkaline picrate solution forms a yellow-orange complex of creatinine picrate which absorbs light at 520 nm, proportional to the amount of creatinine present

• Issues– Subject to interferences from proteins, glucose, uric

acid, medications and others– Enzymatic

• New technology involving coupled reactions

Clearance Measurements

• Evaluation of renal function relies on waste product measurement, specifically the urea and creatinine

• Renal failure must be severe, where only 20% of the nephron is functioning before concentrations of the waste products increase in the blood

• The rate that creatinine and urea are cleared from the body is termed clearance

Clearance

• Definition– Volume of plasma from which a measured

amount of substance can be completely eliminated into urine per unit of time

– Expressed in milliliters per minute

• Function– Estimate the rate of glomerular filtration

Creatinine Clearance

• Used to estimate GFR ( glomerular filtration rate)

• Most sensitive measure of kidney function• Mathematical derivation taking into effect the

serum creatinine concentration to the urine creatinine concentration over a 24- hour period

Creatinine Clearance

Specimen requirements• 24-hour urine

– Keep refrigerated

• Serum/Plasma– Collected during 24-hour

urine collection

Instructions for urine collection

• Empty bladder, discard urine, note exact time

• Collect, save and pool all urine produced in the next 24-hours.

• Exactly 24 hours from start time, empty bladder and add this sample to the collection

Creatinine clearance -

Procedure – Determine creatinine level on serum/plasma - in

mg/dL– Determine creatinine level on 24 hour urine

• measure 24 hr. urine vol. in mL, take a aliquot• make a dilution (usually X 200)• run procedure as for serum• multiply results X dilution factor

– Plug results into formula

Formula

Ucr(mg/dL) X V Ur(mL/24 hour) X 1.73P Cr(mg/dL) X 1440 minutes/ 24 hours A

• U cr= urine creatinine• P cr= serum creatinine• 1.73= normalization factor for body surface

area in square meters• A= actual body surface area

Nomogram1. Left side, find patient’s height( in feet or centimeters)2. On right side, find patient’s weight (lbs or kg)3. Using a straight edge draw a line through the points located4. Read the surface area in square meters, on the middle line

Reference ranges

• Males 97 mL/min- 137 mL/min

• Females 88 mL/min-128 ml/min

Creatinine Clearance Exercise

• Female Patient: 5'6“ & 130 lbs.– Urine Creatinine – 98 mg/dL– Serum Creatinine – 0.9 mg/dL– 24 Hour Urine Volume – 1,200 mL

– Set up calculation

Drawbacks of Creatinine Clearance

• Overestimates the GFR by 10-20%• Timing of serum/urine collection for accurate

analysis• Patients/Health care workers must follow

detailed instructions for proper collection

New Ways to Evaluate eGFR

• Estimates GFR from serum creatinine • Patients age, sex, weight, or race included in

the equation• Common equation used include:

– Modification of Diet in Renal Disease (MDRD)– Cockcroft-Gault– CKD-EPI

Uric acid• Final breakdown product of nucleic acid catabolism - from

both the food we eat, and breakdown of body cells.

• Uric acid is filtered by the glomerulus, majority reabsorbed

• Roles– Assess inherited purine disorders– Confirm diagnosis and treatment of gout– Assist in diagnosis of renal calculi– Prevent uric acid nephropathy during chemotherapy– Detect kidney dysfunction

Clinical Significance: Uric Acid

•Gout

–Increased plasma uric acid–Painful uric acid crystals in joints–Usually in older males ( > 30 years-old )–Associated with alcohol consumption–Uric acid may also form kidney stones

• Other causes of increased uric acid

– Leukemias and lymphomas

» ( DNA catabolism )

– Megaloblastic anemias» ( DNA catabolism )

– Renal disease ( but not very specific )

Specimen Requirements: Uric Acid

• Plasma• Serum• Urine• Serum should be removed from cells ASAP• Avoid lipemia

Uric Acid: Methodology1. Phosphotungstic Acid Reduction — This is the classical chemical method

for uric acid determination. In this reaction, urate reduces phosphotungstic acid to a blue phosphotungstate complex, which is measured spectrophotometrically.

2. Uricase Method — An added enzyme, uricase, catalyzes the oxidation of urate to allantoin, H2O2, and CO2. The serum urate / uric acid may be determined by measuring the absorbance at 293 nm before and after treatment with uricase. (Uricase breaks down uric acid.)

Uric acid + 2H2O + O2 Uricase > Allantoin + H2O2 + CO2 (Absorbs at 293 nm) (Nonabsorbing at 293 nm)

Reference Range: Uric Acid

• Reference values Men 3.5 - 7.2 mg/dL Women 2.6 - 6.0 mg/dL

Other Screening Test for Renal Disease

• Urinalysis– Routine urinalysis good indicator of renal disease

• Microalbumin– Albumin is another sign of renal disease– Usually performed on a random urine

Ammonia

• Formed from the breakdown of amino acids and bacterial metabolism

• Metabolized by the liver• Increases due to renal failure or liver disease

are toxic to the CNS

Specimen Requirements: Ammonia

• Whole blood– EDTA– Heparin

– Patient should not smoke several hours prior to collection, results in contamination

Ammonia: Methodology

1. Glutamate dehydrogenase- enzymatic procedure

2 Oxoglutarate + NH4+ + NADPH Glutamate + NADP+ + H2O

2. NADP + is measured at 340 nm and it is directly proportional to ammonia.

Glutamate dehydrogenase

One final note…• Remember the Renal panel

– Albumin– Glucose– BUN– Creatinine– Calcium– Chloride– Potassium– CO2

– Sodium– Phosphorus

References• Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques,

principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

• Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

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