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 AnalytePlasma Concentration (%) Blood Urea Nitrogen (BUN)45 Amino Acids20 Uric Acid20 Creatinine5 Creatine1-5 Ammonia0.2

4. BUN Blood Urea Nitrogen – Urea is the nitrogenous end-product of protein & AA metabolism. – Urea is formed in the liver when ammonia (NH 3 ) is removed and combined with CO 2. – 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-renal Azotemia  Increased BUN due to non-renal causes  Congestive heart failure, high protein diets, dehydration  Increased Ratio- BUN is high/ creatinine is normal – Renal – Renal Azotemia  Disease directly affects nephron  Glomerulonephritis, Nephrotic syndrome, uremia, etc.  Normal Ratio- both BUN and creatinine are proportionally elevated – Post-renal – Post-renal Azotemia  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 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

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 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

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 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

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 U cr (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 1. 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 located 4. 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, H 2 O 2, and CO 2. 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  Men3.5 - 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 1.Glutamate dehydrogenase- enzymatic procedure 2 Oxoglutarate + NH 4 + + NADPH Glutamate + NADP + + H 2 O 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 – CO 2 – 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. 35