Non-Protein Nitrogen (NPN) Laboratory Procedure PDF

MT 6317 Laboratory Non-Protein Nitrogen (NPN) Faculty of Pharmacy Department of Medical Technology First Term AY 2023-2024 Intended Learning Objectives 1. Describe extensively the different substances found in the body and associate them with diseases found in increasin...

MT 6317 Laboratory Non-Protein Nitrogen (NPN) Faculty of Pharmacy Department of Medical Technology First Term AY 2023-2024 Intended Learning Objectives 1. Describe extensively the different substances found in the body and associate them with diseases found in increasing or decreasing levels; 2. Demonstrate the importance of applying utmost care to patients and allied health professionals; 3. Apply the laboratory protocols, and procedures and confidently release laboratory results to patients and physicians; 4. Explain the principle of different laboratory tests used for Blood Urea Nitrogen determination; 5. Apply proper computation in analysis of Blood Urea Nitrogen determination; and 6. Evaluate the importance of Non-Protein Nitrogenous compounds testing in the diagnosis of diseases. Non-Protein Nitrogen Nitrogen-containing compounds measured after removal of proteins in specimen Measured by converting nitrogen Compound Plasma Urine Concentration to ammonia and subsequent Concentration (% of excreted (% of total NPN) nitrogen) reaction with Nessler’s reagent Urea 45-50 86.0 (K2[Hgl4]) to form a yellow product measured Amino Acids 25 - spectrophotometrically Uric Acid 10 1.7 Creatinine 5 4.5 Specimens: Urine and Plasma Creatine 1-2 - Ammonia 0.2 2.8 Urea Also known as carbamide Highest concentration in blood Produced in the liver from amino groups (-NH2) and free ammonia Major excretory product of protein metabolism Chemical formula: CO(NH2) 2 Urea CLINICAL APPLICATIONS: 1. Evaluation of renal function 2. Assess hydration status 3. Determination of nitrogen balance 4. Aid in the diagnosis of renal disease 5. Verify adequacy of dialysis Blood Urea Nitrogen (BUN) Determination More appropriate term for quantification of urea Conversion of Urea N concentration to urea concentration: Conversion factor (mg/dL to mmol/L): 0.357 Blood Urea Nitrogen (BUN) Determination ENZYMATIC METHOD Most frequently used method Uses urease enzyme to catalyze hydrolysis of urea and the liberated ammonium ion (NH4+) is quantified. Blood Urea Nitrogen (BUN) Determination ENZYMATIC METHOD a. Glutamate dehydrogenase (GLDH)-coupled enzymatic reaction - most common assay - best as a kinetic measurement - measures rate of disappearance of nicotinamide adenine didnucleotide (reduced NADH) at 340 nm b. Indicator dye – used in automated systems, multilayer film reagents and dry reagent strips - measures color change due to change in pH Blood Urea Nitrogen (BUN) Determination ENZYMATIC METHOD c. Conductometric - a specific and rapid test - uses electrode to measure rate of increase in conductivity produced by ammonium ions ISOTOPE DILUTION MASS SPECTROMETRY (IDMS) - reference method - uses isotopically-labeled compound for quantification - detects characteristic fragments following ionization Specimen Considerations Sample: Serum, plasma or urine BLOOD UREA NITROGEN DETERMINATION Enzymatic Reactions Reagents/Materials/Instruments Needed Serologic pipet Urea Reagent Kit: Rubber aspirators 1. Reagent 1: R1 Tris buffer (pH Tissue 7.6), ADP, α-ketoglutarate, Gum label urease, glutamate dehydrogenase, sodium azide Parafilm Specimen (serum or lithium 2. Reagent 2: NADH, sodium heparinized plasma) azide (irritates eyes and skin) Spectrophotometer 3. Standard Urea: 50 mg/dL or Kahn test tubes 8.33 mmol/L (may vary depending on manufacturer) Centrifuge Procedure 1. Prepare and label test tubes properly. 2. Prepare the working reagent by mixing 1 volume of reagent with 1 volume of reagent 2 into a separate tube or vessel. 3. Follow the pipetting scheme. 4. Deliver the volume of the sample first by touching the tip of the pipet at the bottom of the tube Pipet into cuvet Blank Std Cn Cp Unk Standard Solution - 0.01 mL - - - Control Normal Serum - - 0.01 mL - - Control Pathologic Serum - - - 0.01 mL - Patient’s serum - - - - 0.01 mL Reagent 1A 1.0 mL 1.0 mL 1.0 mL 1.0 mL 1.0 mL Procedure 5. Set up the spectrophotometer at 340 nm. 6. Add the reagent by touching the tip of the pipet to the side of the tube. 7. Mix the solution by covering the mouth of the tube with a parafilm and invert the tubes 2-3 times. 8. Start incubation time 9. Feed the solution in the machine at 27 seconds, read and record absorbance as A1. 10.Continue the timer and at exactly 90 seconds, read and record again the absorbance as A2. Assay Requirements Wavelength: 340 nm Optical path: 1 cm Temperature: 37 ͦ C Read against distilled water Calculations Conversion Factor: C (BUN) = 0.466 x C (Urea) C (Urea) = 2.14 x C (BUN) Reference Intervals Urea Nitrogen (Adults) Conventional Unit International Unit Plasma or Serum 6-20 mg/dL 2.1-7.1 mmol/L 24-hr urine 12-20 g/day 0.43-0.71 mmol/day Conversion factor (mg/dL to mmol/L): 0.357 Clinical Significance Azotemia Elevated urea concentration in blood Uremia/Uremic Syndrome Increased urea level accompanied by renal failure Ellitrol Assayed Chemistry Control Levels 1 and 2 LOT No. 123456 EXP: 12/2023 Method Unit Level 1 N123456 Level 2 P123456 Mean Range Mean Range Blood Urea Nitrogen Urease (UV Kinetic) mg/dL 15.5 13.3-17.7 47.0 42.4-51.6 Indophenol mg/dL 33.2 28.5-37.9 101.0 90.7-111.0 References Bishop, M.L., et al. (2022). Clinical Chemistry: Principles, Techniques and Correlations(9th ed.). Wolters Kluwer. McPherson, R. A. & Pincus, M.A. (2017). Henry’s Clinical Diagnosis and Management by Laboratory Methods (23rd ed.). Elsevier, Inc. Cortel, M.R.B., Asuncion, V.D. (2023). Worktext in Clinical Chemistry 1 and 2. C&E Publishing, Inc.