Chemical analysis of Urine DrMohamed Mahmoud Nour Eldein

Chemical analysis of Urine Dr/Mohamed Mahmoud Nour Eldein Ph. D Biochemistry n Assistant Professor of Biochemistry Faculty of Medicine Umm AL-Qura University

Chemical Analysis

Urine dipsticks (Reagent Strips) n Urine dipstick are plastic strips on which are attached to a series of chemically impregnated absorbent pads, each pad contain certain chemicals that react with substance in the urine producing a color change in pad, this color change is compared with a series of known standards.

Chemical Analysis Urine Dipstick Glucose Bilirubin Ketones Specific Gravity Blood p. H Protein Urobilinogen Nitrite Leukocyte Esterase

Reagent Strips

Procedure n Reagent strips are used only once and discarded. n Testing n Perform within 1 hour after collection n Allow refrigerated specimens to return to room temperature. n Dip strip briefly, but completely into well mixed, room temperature urine sample. n Withdraw strip. n Blot briefly on its side. n Keep the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.

Procedure n Instruments are available which detect color changes electronically and prints out results

Handling and Storage of Strips n Handling and Storage n Keep strips in original container n Do not touch reagent pad areas n Reagents and strips must be stored properly to retain activity n n n Protect from moisture and volatile fumes Stored at room temperature Use before expiration date

Sources of Error n Timing - Failure to observe color changes at appropriate time intervals may cause inaccurate results. n Lighting - Observe color changes and color charts under good lighting. n QC - Reagent strips should be tested with positive controls on each day of use to ensure proper reactivity. n Sample - Proper collection and storage of urine is necessary to insure preservation of chemical.

Sources of Error n Testing cold specimens - would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing n Inadequate mixing of specimen - could result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dipping n Over-dipping of reagent strip - will result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine

The Urine Dipstick: Negative Trace (100 mg/d. L) Glucose Chemical Principle Glucose Oxidase + (250 mg/d. L) Glucose + 2 H 2 O + O 2 ---> Gluconic Acid + 2 H 2 O 2 ++ (500 mg/d. L) Horseradish Peroxidase +++ (1000 mg/d. L) ++++ (2000+ mg/d. L) 3 H 2 O 2 + KI ---> KIO 3 + 3 H 2 O Read at 30 seconds RR: Negative

Uses and Limitations of Urine Glucose Detection Significance n n Diabetes mellitus. Renal glycosuria. Limitations n n n Interference: reducing agents, ketones. Only measures glucose and not other sugars. Renal threshold must be passed in order for glucose to spill into the urine. Other Tests n Cu. SO 4 test for reducing sugars.

Urinalysis Glucose Result Urine versus Blood Glucose ++ + trace Negative 200 400 600 800 Blood Glucose (mg/d. L) 1000

The Urine Dipstick: Bilirrubin Negative Chemical Principle + (weak) Acidic Azobilirubin Bilirubin + Diazo salt -----> ++ (moderate) +++ (strong) Read at 30 seconds RR: Negative

Bilirubin n Bilirubin is a byproduct of the breakdown of hemoglobin. n Normally contains no bilirubin. n Presence may be an indication of liver disease, bile duct obstruction or hepatitis. n Since the bilirubin in samples is sensitive to light, exposure of the urine samples to light for a long period of time may result in a false negative test result.

Ketones n Ketones are excreted when the body metabolizes fats incompletely (ketonuria)

The Urine Dipstick: Negative Ketones Chemical Principle Trace (5 mg/d. L) + (15 mg/d. L) ++ (40 mg/d. L) +++ (80 mg/d. L) ++++ (160+ mg/d. L) Acetoacetic Acid + Nitroprusside ------> Colored Complex Read at 40 seconds RR: Negative

Uses and Limitations of Urine Ketone Detection Significance - Diabetic ketoacidosis - Prolonged fasting Limitations - Interference: expired reagents (degradation with exposure to moisture in air) - Only measures acetoacetate not other ketone bodies (such as in rebound ketosis). Other Tests - Ketostix (more sensitive tablet version of same assay) - Serum glucose measurement to confirm DKA

Specific gravity n Depends on the concentration of various solutes in the urine. n Specific gravity reflects kidney's ability to concentrate. n Want concentrated urine for accurate testing, best is first morning sample. n Low – specimen not concentrated, kidney disease. n High – first morning, certain drugs n Measured by-urinometer - refractometer - dipsticks

Urinometer n Take 2/3 of urinometer container with urine n Allow the urinometer to float into the urine n Read the graduation at the lowest level of urinary meniscus n Correction of temperature & albumin is a must. n Urinometer is calibrated at 15 or 200 c So for every 3 oc increase/decrease add/subtract 0. 001 For 1 gm/dl of albumin add 0. 001

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The Urine Dipstick: Specific Gravity 1. 000 1. 005 1. 010 1. 015 1. 020 1. 025 1. 030 Chemical Principle X+ + Polymethyl vinyl ether / maleic anhydride --------> X+-Polymethyl vinyl ether / maleic anhydride + H+ H+ interacts with a Bromthymol Blue indicator to form a colored complex. Read up to 2 minutes RR: 1. 003 -1. 035

Uses and Limitations of Urine Specific Gravity Significance - Diabetes insipidus Limitations - Interference: alkaline urine - Does not measure non-ionized solutes (e. g. glucose) Other Tests - Refractometry - Hydrometer - Osmolality measurement (typically used with water deprivation test)

High specific gravity(hyperosthenuria) n Normal-1. 016 -1. 022 n Causes All causes of oliguria Glycosuria

Low specific gravity(hyposthenuria) n All causes of polyuria except glycosuria n Fixed specific gravity (isosthenuria)=1. 010 Seen in chronic renal disease when kidney has lost the ability to concentrate or dilute

Blood n Presence of blood may indicate infection, trauma to the urinary tract or bleeding in the kidneys. n False positive readings most often due to contamination with menstrual blood.

The Urine Dipstick: Negative Trace (non-hemolyzed) Moderate (non-hemolyzed) Trace (hemolyzed) + (weak) ++ (moderate) +++ (strong) Blood Chemical Principle Lysing agent to lyse red blood cells Diisopropylbenzene dihydroperoxide + Tetramethylbenzidine Heme ------> Colored Complex Read at 60 seconds RR: Negative Analytic Sensitivity: 10 RBCs

Uses and Limitations of Urine Blood Detection Significance - Hematuria (nephritis, trauma, etc) - Hemoglobinuria (hemolysis, etc) - Myoglobinuria (rhabdomyolysis, etc) Limitations - Interference: reducing agents, microbial peroxidases - Cannot distinguish between the above disease processes Other Tests - Urine microscopic examination - Urine cytology

Urinary p. H/ reaction n Reaction reflects ability of kidney to maintain normal hydrogen ion concentration in plasma & ECF n Normal= 4. 6 -8 n Tested by- 1. litmus paper 2. p. H paper 3. dipsticks

The Urine Dipstick: p. H 5. 0 6. 5 7. 0 7. 5 8. 0 8. 5 Chemical Principle H+ interacts with: Methyl Red (at high concentration; low p. H) and Bromthymol Blue (at low concentration; high p. H), to form a colored complexes (dual indicator system) Read up to 2 minutes R. R. : 4. 5 -8. 0

Acidic urine n Ketosis-diabetes, starvation, fever n Systemic acidosis n UTI- E. coli n Acidification therapy

Alkaline urine n Strict vegetarian n Systemic alkalosis n UTI- Proteus n Alkalization therapy

Uses and Limitations of Urine p. H Detection Significance - Acidic (less than 4. 5): metabolic acidosis, high-protein diet - Alkaline (greater than 8. 0): renal tubular acidosis (>5. 5) Limitations - Interference: bacterial overgrowth (alkaline or acidic), “run over effect” effect of protein pad on p. H indicator pad Other Tests - Titrable acidity - Blood gases to determine acid-base status

p. H Run Over Effect Glucose Bilirubin Ketones Specific Gravity Blood p. H Protein Urobilinogen Nitrite Leukocyte Esterase Buffers from the protein area of the strip (p. H 3. 0) spill over to the p. H area of the strip and make the p. H of the sample appear more acidic than it really is.

Protein n Presence of protein (proteinuria) is an important indicator of renal disease. n False negatives can occur in alkaline or dilute urine or when primary protein is not albumin.

The Urine Dipstick: Negative Trace + (30 mg/d. L) Protein Chemical Principle “Protein Error of Indicators Method” Pr H Pr Pr H H Pr Pr Pr H + Tetrabromphenol Blue + ++ (100 mg/d. L) H+ H H (buffered to p. H 3. 0) + + H H +++ (300 mg/d. L) Pr Pr Pr ++++ (2000 mg/d. L) Pr Read at 60 seconds RR: Negative

Causes of Proteinuria Functional - Severe muscular exertion - Pregnancy - Orthostatic proteinuria Pre-Renal - Fever - Renal hypoxia - Hypertension Renal - Glomerulonephritis - Nephrotic syndrome - Renal tumor or infection Post-Renal - Cystitis - Urethritis or prostatitis - Contamination with vaginal secretions

Uses and Limitations of Urine Protein Detection Significance - Proteinuria and the nephrotic syndrome. Limitations - Interference: highly alkaline urine. - Much more sensitive to albumin than other proteins (e. g. , immunoglobulin light chains). Other Tests - Sulfosalicylic acid (SSA) turbidity test. - Urine protein electrophoresis (UPEP) - Bence Jones protein

Urobilinogen n Urobilinogen is a degradation product of bilirubin formed by intestinal bacteria. n It may be increased in hepatic disease or hemolytic disease

The Urine Dipstick: 0. 2 mg/d. L 1 mg/d. L Urobilinogen Chemical Principle Urobilinogen + Diethylaminobenzaldehyde (Ehrlich’s Reagent) 2 mg/d. L 4 mg/d. L 8 mg/d. L -------> Colored Complex Read at 60 seconds RR: 0. 02 -1. 0 mg/d. L

Uses and Limitations of Urobilinogen Detection Significance - High: increased hepatic processing of bilirubin - Low: bile obstruction Limitations - Interference: prolonged exposure of specimen to oxygen (urobilinogen ---> urobilin) - Cannot detect low levels of urobilinogen Other Tests - Serum total and direct bilirubin

Nitrite n Nitrite formed by gram negative bacteria converting urinary nitrate to nitrite

The Urine Dipstick: Nitrite Chemical Principle Negative Positive Acidic Nitrite + p-arsenilic acid -------> Diazo compound + Tetrahydrobenzoquinol -----> Colored Complex Read at 60 seconds RR: Negative

Uses and Limitations of Nitrite Detection Significance - Gram negative bacteriuria Limitations - Interference: bacterial overgrowth - Only able to detect bacteria that reduce nitrate to nitrite Other Tests - Correlate with leukocyte esterase and - Urine microscopic examination (bacteria) - Urine culture

Leukocytes n Leukocytes (white blood cells) usually indicate infection. n Leucocyte esterase activity is due to presence of WBCs in urine while nitrites strongly suggest bacteriuria.

The Urine Dipstick: Leukocyte Esterase Chemical Principle Negative Trace + (weak) ++ (moderate) +++ (strong) Derivatized pyrrole amino acid ester Esterases ------> 3 -hydroxy-5 -phenyl pyrrole + diazo salt -------> Colored Complex Read at 2 minutes RR: Negative Analytic Sensitivity: 3 -5 WBCs

Uses and Limitations of Leukocyte Esterase Detection Significance - Pyuria - Acute inflammation - Renal calculus Limitations - Interference: oxidizing agents, menstrual contamination Other Tests - Urine microscopic examination (WBCs and bacteria) - Urine culture

Normal Values n Negative results for glucose, ketones, bilirubin, nitrites, leukocyte esterase and blood. n Protein negative or trace. n p. H 5. 5 -8. 0 n Urobilinogen 0. 2 -1. 0 Ehrlich units