KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (Bioc 416) 2012 T.A Nouf Alshareef

(96%) water Inorganic: Cl -, Na, K. trace amounts of: sulfate, HCO 3 etc.) Urine: Is an ultrafiltrate of plasma from which glucose, amino acids, water and other substances essential to body metabolism have been reabsorbed. Urine carries waste products and excess water out of the body. (4%) dissolved solids: (2%) Urea: ( half) (2%) Other compounds Organic: creatinine uric acid

Factors affect on urine constituents: dietary intake, physical activity, body metabolism, endocrine function others.

Urine Analysis: Routine Urinalysis (Routine-UA): It consists of a group of tests performed as part of physical examination. It involves macroscopic and microscopic analysis. Type of analysis: macroscopic analysis: microscopic examination: urine sediment is examined under microscope to identify the components of the urinary sediments. physical characteristics chemical analysis

Steps in basic urine analysis Three steps analysis: First: physical characteristics of urine are noted and recorded. Second: series of chemical tests is run. Third: urine sediment is examined under microscope to identify the components of sediments.

Urine Collection: Types of urine specimens: type of specimen and collection procedure are determined by physician and depend on the tests to be performed. There are basically four types of urine specimens: 1.First morning specimen 2.Random urine specimen 3.Fractional collection 4.Timed collection Composition and concentration of urine changes during 24hr Urine conc. vary according: to water intake and physical activities.

Truly representative sample: it is necessary to regulate: time of collection, length of collection period, patient's dietary, medical intake and method of collection. Initial morning sample is preferred (particularly for protein analysis) because they are more concentrated from overnight retention in bladder. Time of analysis: - must analyzed within 1h at room temp. or within 8hr at 2 o C- 8 o C - If not assayed within these time limits, several changes will occur. sample should collected in a clean container. urine container must be sterile if the urine is to be cultured. For microscopic examination, the urine must be fresh.

direct visual observation. Normal fresh urine: Color: pale or dark yellow-amber, clear. Vol: ml/24hr. Physical examination involves: 1.Color 2.Transparency 3.Odour 4.Volume 5.pH 6.Specific gravity

1- Color: Color intensity of urine correlates to concentration. Darker color means more concentrated sample. Urochrome Amber yellow Urochrome (derivative of urobilin, produce from bilirubin degradation, is pigment found in normal urine). Colorless due to reduced concentration. Silver or milky appearance Pus, bacteria or epithelial cells Reddish brown Blood (Hemoglobin). Yellow foam Bile or medications. Orange, green, blue or red medications.

2- Transparency: Is classified as clear or turbid. In normal urine: the main cause of cloudiness is crystals and epithelial cells. In pathological urine: it is due to pus, blood and bacteria. Degree of cloudiness depends on: pH and dissolved solids  Turbidity: may be due to gross bacteriuria,  Smoky appearance: is seen in hematouria.  Thread-like cloudiness: is seen in sample full of mucus.

3- Odour: Odour has little diagnostic significance. 1.Aromatic odour------> Normal urine due to aromatic acids. 2.Ammonia odour------> On standing due to decomposition of urea. 3.Fruity odour > Diabetes due to the presence of ketones. 4- Volume: Is important part of assessment for fluid balance and kidney functions. Adults produce from 750ml-2500ml / 24h, with the average of about 1.5L per person. For RUA, a 10ml-12ml of sample is optimal for accurate of analysis

5- pH: pH measure acidicity or alkalinity (basic) of urine Normal urine pH: Increased acidity in urine: due to diabetes or medications. Urine sample must be fresh (why?) ( on standing urine become alkaline as a result of ammonia liberation due to urea decomposition). 6- Specific Gravity (SG): measures the amount of substances dissolved in urine. also indicates how well kidneys are able to adjust amount of water in urine. higher SG: more solid material is dissolved in urine

Organic: urea, uric acid, creatinine Inorganic: Cl -, PO 4 -3, HBO 3, NH 4, SO Urea: 1ml urine + 3ml NaOCL (sodium hypochlorite) ==>Evolution of N2 gas. 2- Uric acid UA: 1ml urine ml 10% NaOH +1ml Folins reagent ===> Blue color. 3- Creatinine: - 1ml urine + drops Picric acid + drops NaOH ====> red color ppt. Note: if reaction is acidified with HCL, the color changes to yellow.

4- Chloride: -1ml urine + drops HNO 3 +1 ml AgNO 3 ===> white ppt of AgCL 5- Phosphate: 1ml urine + 1ml conc. HNO 3 + 1ml NH 4 -molybdate===>Yellow color. 6- Carbonate: 1ml urine + drops conc. HCL ==> Na 2 CO HCL ==> H 2 O + 2NaCL + CO 2 7- Ammonia: - Make urine alkaline with NaOH. Close the tube with a cork containing another side tube dipped in Nessler's reagent. Heat the urine and then notice the evolving of NH3 in Nessler's reagent - Detect NH3 by its odour. - 1ml urine + 1ml phenol + 1ml NaBr =======> Blue color. 8- Sulphates: - 1ml urine + 2 drops conc. HCL + few drops BaCL2 ===> White ppt of BaSO4. SO4 + BaCL2 =====> BaSO4 + 2CL- (effervescence)

Lab Practices: Collect urine in a clean container. Run routine UA on the sample by using both urine strip and the method described before for chemical analysis. Record the results in the lab report of UA.