Urea (UV) Test Procedure PDF - BEACON Diagnostics

# LIQUIZYME UREA (UV) (UV GLDH METHOD) ## Intended Use: The reagent kit is intended for the "in vitro" quantitative determination of Urea in serum, Plasma and Urine. ## Clinical Significance: Urea is the end product of the protein metabolism. It is synthesized in the liver from the ammonia prod...

# LIQUIZYME UREA (UV) (UV GLDH METHOD) ## Intended Use: The reagent kit is intended for the "in vitro" quantitative determination of Urea in serum, Plasma and Urine. ## Clinical Significance: Urea is the end product of the protein metabolism. It is synthesized in the liver from the ammonia produced by the catabolism of amino acids. It is transported by the blood to the kidneys from where it is excreted. Increased levels are found in renal diseases, urinary obstructions, shock, congestive heart failure and burns. Decreased levels are found in liver failure and pregnancy. ## Principle: Urease hydrolyzes urea to ammonia and CO2. The ammonia formed further combines with a Ketoglutarate and NADH to form Glutamate and NAD. The rate of oxidation of NADH to NAD is measured as a decrease in absorbance in a fixed time, which is proportional to the urea concentration in the sample. ## Reaction: ``` Urease Urea + H2O + 2H -> 2NH4+ + CO2 GLDH 2a-ketoglutarate + 2NH4+ + 2NADH -> 2L-Glutamate + 2NAD+ + 2H2O ``` ## Contents: - Reagent 1: Urea Enzyme Reagent - Reagent 2: Urea Substrate Reagent - Reagent 3: Urea Standard 50 mg/dl ## Materials Required But Not Provided: - Clean & Dry Glassware. - Laboratory Glass Pipettes or Micropipettes & Tips. - Bio-Chemistry Analyzer. ## Samples: Serum, plasma, urine. Do not use anticoagulants containing fluoride or ammonium ions. Dilute urine 1+49 with distilled water and multiply results by 50. ## Preparation of Working Reagent & Stability: Working reagent: Mix 4 parts of Reagent 1 + 1 part of Reagent 2. Working reagent is stable for 5 days at 15-25 °C and for 4 weeks at 2-8 °C. Unopened kit is stable til expiry date mentioned on the kit when stored at 2-8 °C. Do not freeze the reagents. ## General System Parameters: | Parameter | Value | |---|---| | Reaction Type | Fix Time (Decreasing) | | Wave Length | 340 nm. | | Cuvette Temp | 37°C | | Delay Time | 30 Sec. | | Interval Time | 60 Sec. | | No. of reading | 1 | | Reagent Volume | 1.0 mL | | Sample Volume | 10 μL | | Standard | 50 mg/dl | | Zero Setting | Deionised water | | Light Path | 1 cm. | ## Procedure: Pipette into clean dry test tube labeled as Standard (S) and Test (T): | Addition sequence | S | T | |---|---|---| | Working reagent | 1 ml | 1 ml | | Standard | 10µl | - | | Sample | - | 10 μl | Mix well and read the initial absorbance A for the Standard and the Test after exactly 30 seconds. Read another absorbance A of the Standard and the Test exactly 60 seconds later. Calculate the change in absorbance ΔA for both the Standard and Test. For Standard Δ AS = AS2 - AS1 For Test Δ AT = AT2 - AT1 ## Calculation: Urea (mg/dl) = Δ AT x 50 / Δ AS ## Normal Value: - Serum / Plasma: 10-40 mg/dl - Urine: 26 to 43 g/24 hrs. ## Urea/Creatinine Ratio: - 25:40 [(mmol/L)/(mmol/L)] - 20:35 [(mg/dL)/(mg/dL)] Each Laboratory should establish it's own normal range representing its patient population. ## Linearity: The procedure is linear upto 300 mg/dl. If the value exceed this limit, dilute the serum with normal saline (NaCl 0.9%) and repeat the assay. Multiply result by dilution factor. ## Quality Control: For accuracy it is necessary to run known controls with every assay. ## Limitation & Precautions: The reagents should be handled with caution, avoiding swallowing and contact with skin, eyes and mucous membranes. The use of the laboratory reagents according to good laboratory practice is recommended. ## References: 1. Thomas L. Clinical Laboratory Diagnostics. 1st ed. Frankfurt: TH-Books Verlagsgesellschaft; 1998 p.374-7. 2. Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3 rd ed. Philadelphia: W.B Saunders Company; 1999. p. 1838. 3. Talke H, Schubert GE. Enzymatische Harnstoff-bestimmung in Blut und Serum im optischen Test nach Warburg (Enzymatic determination of urea in blood and serum with the optical test according to Warburg). Klin Wschr 1965; 43:174-5. ## Pack size: | Code No. | Reagent 1 | Reagent 2 | Reagent 3 | |---|---|---|---| | S16C | 2x20 ml | 1x10 ml | 3.0 ml | | S16A | 4x20 ml | 2x10 ml | 3.0 ml | # BEACON DIAGNOSTICS PVT. LTD 424, NEW GIDC, KABILPORE, NAVSARI - 396 424. INDIA