MLAB 2401: Clinical Chemistry

1. MLAB 2401: Clinical Chemistry Renal Assessment

2. 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

3. Clinically Significant NPN’s

4. 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

5. 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

6. BUN / Creatinine Ratio • Normal • BUN / Creatinine ratio is 12 – 20 to 1 • Pre-renalAzotemia • Increased BUN due to non-renal causes • Congestive heart failure, high protein diets, dehydration • Increased Ratio- BUN is high/ creatinine is normal • RenalAzotemia • Disease directly affects nephron • Glomerulonephritis, Nephrotic syndrome, uremia, etc. • Normal Ratio- both BUN and creatinine are proportionally elevated • Post-renalAzotemia • Occurs after urine has left the kidney- due to obstruction • Increased Ratio- BUN is high • Plasma creatinine also elevated

7. Specimen Requirements: BUN • Plasma • Serum • 24-hour Urine • nonhemolyzed

8. 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

9. 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

10. Advantages of Creatinine • Formed at a constant rate • Readily excreted • Not reabsorbed • Not affected by diet

11. Reference Range/Significance: Creatinine Significance Reference Range Urine 0.8-2.0gm/ 24 hour Serum 0.5-1.5mg/dL • Evaluates renal function • Follows progress of renal disease • Increased results • Renal disease • Decrease in GFR • Obstruction in urinary system • Decreased muscle mass

12. Specimen requirements: Creatinine • Plasma • Serum • Urine ( 24 hour or random) • Avoid hemolysis • Avoid icterus

13. 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 creatininepicrate 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

14. 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

15. 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

16. 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

17. Creatinine Clearance Specimen requirements 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 • 24-hour urine • Keep refrigerated • Serum/Plasma • Collected during 24-hour urine collection

18. 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

19. Formula Ucr(mg/dL) X V Ur(mL/24 hour)X 1.73 P 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

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

21. Reference ranges • Males • 97 mL/min- 137 mL/min • Females • 88 mL/min-128 ml/min

22. 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

23. 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

24. 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

25. 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

26. 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 )

27. Specimen Requirements: Uric Acid • Plasma • Serum • Urine • Serum should be removed from cells ASAP • Avoid lipemia

28. Uric Acid: Methodology 1. 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)

29. Reference Range: Uric Acid • Reference values • Men 3.5 - 7.2 mg/dL • Women 2.6 - 6.0 mg/dL

30. 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

31. 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

32. Specimen Requirements: Ammonia • Whole blood • EDTA • Heparin • Patient should not smoke several hours prior to collection, results in contamination

33. Ammonia: Methodology • 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

34. One final note… • Remember the Renal panel • Albumin • Glucose • BUN • Creatinine • Calcium • Chloride • Potassium • CO2 • Sodium • Phosphorus

35. 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 .