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Chapter 4:
Chemical Analysis of Urine
Graff’s Textbook of Urinalysis and Body Fluids,
Second Edition
Mundt, L. & Shanahan K.
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
Chapter Outline
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Introduction to reagent strip testing
pH
Protein
Glucose
Ketones
Blood
Bilirubin
Urobilinogen
Nitrite
Leukocyte esterase
Additional parameters
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Introduction to reagent strip testing
Illustration of Multistix 10 SG
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Introduction to reagent strip testing
(cont.)
• Dipping method
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Introduction to reagent strip testing
(cont.)
• Reading method
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Introduction to reagent strip testing
(cont.)
• For accurate results:
– Store reagent strips in their bottle with lid closed at room
temperature.
– Do not expose strips to
• Sunlight
• Heat
• Cold
• Volatile substances
• Moisture (each bottle contains a desiccant)
– Only remove the number of strips you need for immediate
testing.
– Do not use strips that have discolored pads.
– Do not use expired reagent strips.
– Test urine which is brought to room temperature.
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Introduction to reagent strip testing
(cont.)
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Urine pH
• Kidneys regulate acid-base balance
• pH changes demonstrate and assess this
function
• Must be evaluated in clinical context
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Urine pH
pH color chart
• Acid indicator – methyl red
• Alkaline indicator – bromthymol blue
• Read 60 seconds after dipping
• pH ranges from 5.0 to 8.5 in half units
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Urine pH
• pH
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Urine pH: Significance
• Persistent Acidity
– Acidifying drugs
– Dehydration
– Diabetes
– Diarrhea
– Fever
– Gout
– High protein diets
– Pulmonary emphysema
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Urine pH: Significance (cont.)
• Persistent alkalinity
– Alkaline drugs
– Acute and chronic renal failure
– Diuretics
– Renal tubular acidosis
– Urinary tract infections
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Urine protein
• Small amounts of low–molecular weight protein are
filtered at the glomerulus
• Most of this protein is reabsorbed in the tubules
• Less than 150 mg/24 h (or 20mg/dL) is excreted
• Mucoprotein Tamm–Horsfall is secreted by the
renal tubules is also excreted
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Urine protein (cont.)
Protein color chart
• Principle: “protein error of indicators”
• Tetrabromphenol blue most common indicator
• Color ranges from yellow to blue
• Detects primarily albumin
• Negative does not rule out other significant proteins
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Urine protein: false positive
• Highly buffered alkaline urine (medications or old urine)
• Prolonged exposure to the sample
• Container cleaning compounds (quaternary ammonia)
• Phenazopyridine
• Plasma expander polyvinylpyrrolidon
• Chlorhexidine gluconate (skin cleansers)
• Blood
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Urine protein: false negative
• Dilute urines
• Elevated amounts of proteins other than albumin
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Urine protein: significance
• Physiology causes
• Exercise
• Emotional stress
• Exposure to heat or cold
• Fever
• Pregnancy
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Urine protein: significance
• Pathologic causes
• Glomerular nephritis
• Pyelonephritis
• Malignant hypertension
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Urine glucose
• Normally contained in glomerular ultrafiltrate
• Reabsorbed in the proximal tubule
• Usually not present unless threshold levels exceeded
• 160-180 mg/dl
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Urine glucose
Glucose color chart
Reaction A:
glucose oxidase
Glucose + O2
gluconic acid + H2O2
Reaction B:
peroxidase
H2O2 + chromogen
oxidized chromogen
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Urine glucose: false positive
• Oxidizing cleaning agents
• Peroxide
• Hypochorite
• Elevated urobiliongen with some automated methods
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Urine glucose: false negative
• Decreased sensitivity in:
• Cool urine
• Urine with high specific gravity
• Alkaline urine
• High ketone levels
• Ascorbic acid (Vitamin C) high doses
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Urine with reducing substances
• Other sugars
• Fructose
• Galactose
• Lactose
• Maltose
• Dextrins
• Homogentisic acid
• Glucuronates
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Clinitest
Clinitest color chart
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Clinitest: false positive
• Ascorbic acid
• Nalidixic acid
• Cephalosporins
• Probenecid
• Urinary preservatives:
– formalin
– formaldehyde
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Clinitest: false negative
• Technique errors
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Urine glucose: significance
• Normal renal threshold – 160-189 mg/dl
• Glycosuria dependent on:
• Blood glucose levels
• Glomerular filtration rate
• Tubular reabsorption
• Seen in diabetes and congenital forms of glucosuria
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Urine reducing sugars: significance
• Routinely performed on urine from children 2 years
old and younger
• Early detection of galactosemia
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Urine ketones: normal formation
• Ketones result from the breakdown of fat.
• One of the intermediate products of fatty acid
• Breakdown is acetyl CoA.
• Acetyl CoA enters the citric acid cycle (Krebs cycle) in
the body if fat and carbohydrate degradation are
appropriately balanced.
• The first step in the Krebs cycle is the reaction of
acetyl CoA with oxaloacetate to yield citrate.
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Urine ketones: excessive formation
• Oxaloacetate will be used to form glucose when
carbohydrates are absent or improperly used.
• No oxaloacetate available for condensation with acetyl
CoA.
• CoA cannot enter the Krebs cycle and is diverted to
the formation of ketone bodies.
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Urine ketones
O
||
CO2
CH3-C-CH3
O
(Acetone)
||
H3C-C-CH2-COOH
OH
||
(Acetoacetic acid)
NADH2
CH3-CH-CH2-COOH
(β-hydroxybutyric acid)
NAD+
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Urine ketones
Ketone color chart
alkaline pH
Acetoacetic acid + Na nitroprusside + Glycine
violet–purple color
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Urine ketone: false positive or atypical color
• Highly pigmented urines
• Combination of high specific gravity and a low pH
• Levodopa metabolites
• Sulfhydryl groups
• Phenylketones
• Phthalein compounds
• Positive and questionable results may be confirmed with
a tablet test.
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Urine ketone: false negative
• Controls solutions that use acetone.
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Urine ketones confirmatory test
Acetest color chart
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Urine ketone
• Ketone blood level are normally very low (2-4 mg/dl).
– 20% acetoacetic acid
– 2% acetone
– 78% B-hydroxybutyric acid.
• Acetone is lost into the air at room temperature
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Urine ketone: significance
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Diabetes mellitus
Diarrhea
Exposure to cold
Fasting
Fever
Insufficient carbohydrate intake
Malnutrition
Strenuous exercise
Vomiting
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Urine blood/hemoglobin
• Hematuria
• Glomerulus
• Renal tubules
• Ureters
• Bladder
• Hemoglobinuria
• Glomerular filtrate
• Myoglobinuria
• Glomerular filtrate
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Urine blood/hemoglobin
Blood color chart
hemoglobin
H2O2 + chromogen
oxidized chromogen+ H2O2
peroxide
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Urine blood/hemoglobin: false positive
• Oxidizing contaminants
• Hypochlorites
• Bacterial peroxidases
• Menstrual blood
• Povidoneiodine (Betadine)
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Urine blood/hemoglobin: false negative
• Technique errors inadequate mixing
• Formalin preservative
• High concentrations of ascorbic acid (Vitamin C)
• Captoprin (Capoten)
• High specific gravity
• Nitrites
• Proteins
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Urine blood/hemoglobin: significance
• Hematuria
• Hemoglobinuria
• Myoglobinuria
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Urine blood/hemoglobin: hematuria
• Intact RBCs
• If lysed in urine, “ghosts” may be present in sediment
• Urine may appear normal with small amounts
• Urine is red with greater amounts of blood
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Urine blood/hemoglobin: hematuria
• Can indicate renal diseases
• Glomerular
• Tubular
• Interstitial
• Vascular
• Also present in patients with
• Lithiasis (kidney stones)
• Urinary tract infections
• Urinary tract tumors
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Urine blood/hemoglobin: hemoglobinuria
• Free hemoglobin
• Intervascular hemolysis
• Incompatible blood transfusions
• Hemolytic anemia
• Burns
• Cold auto-immune anemia
• Electric shock
• Lysis only in urine does not carry the same significance
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Urine blood/hemoglobin: myoglobinuria
• Damage to cardiac or skeletal muscle
• Crush injuries
• Electric shock
• Myocardial infarct
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Urine blood/hemoglobin vs myoglobin
• Both will produce positive reagent strip blood results
• Marcoscopic differentiation
• red plasma plus red urine equals hemoglobin
• clear plasma plus red urine equals myoglobin
• Chemical differentiation
• Ammonium sulfate test
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Urine bilirubin formation
• Formed by hemoglobin degradation
• bound to albumin and transported through the blood to
the liver.
• unconjugated bilirubin is insoluble in water and cannot
be filtered through the glomerulus.
• conjugated with glucuronic acid in the liver to form
bilirubin diglucuronide.
• Conjugated bilirubin (direct) is water soluble and is
excreted by the liver through the bile duct and into
the duodenum.
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Urine bilirubin
• small amounts of conjugated bilirubin regurgitate back
from the bile duct and into the blood system.
• filtered through the glomerulus and excreted in the
urine whenever the plasma level is increased.
• Normally, no detectable amounts are present in urine
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Urine bilirubin
Bilirubin color chart
acid
Bilirubin + diazide
azobilirubin
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Urine bilirubin: false positive
• Technique errors
– Reading after the prescribed time
• atypical color reactions produced by:
– Indican
– Metabolites of etodolac (Lodine)
– Chlorpromazine (Thorazine)
– Metabolites of phenazopyridine
• Confirm results with Ictotest
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Urine bilirubin: false negative
• Large amounts of ascorbic acid decrease the sensitivity
• High levels of nitrite
• Exposure to light and room temperature
– Bilrubin oxidizes to biliverdin
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Urine bilirubin confirmatory test
Ictotest color reactions.
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Urine bilirubin: significance
• Normally, no bilirubin is present in urine
• Present in urine:
– when bile flow to colon is obstructed
– liver damage
– Hepatitis
– cholestasis
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Urine urobilinogen formation
• Formed from bilirubin in the intestines
• Urobilinogen (colorless) oxidizes to urobilin (brown)
• Most lost in the feces
• About 10–15% reabsorbed into the bloodstream,
returns to the liver, and reexcreted into the intestines
• A small amount of this urobilinogen is excreted by the
kidneys into the urine
• Normal level: 1–4 mg/24 h or less than 1.0 Ehrlich unit
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Urine urobilinogen
Urobilinogen color chart
p-Dimethylaminobenzaldehyde + urobiligen = azo dye
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Urine urobilinogen: false positive
• p-aminosalicylic acid
• sulfonamides,
• p-aminobenzoic
• Phenazopyridine
• Prophobilionogen
– May use Watson-Shwartz test to differeniate
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Urine urobilinogen: false decrease
• A true absence of urobilinogen is not detectable.
• Broadspectrum antibiotics
• Nitrite
• Formalin
• Improper storage of specimen
–
oxidation of urobilinogen to urobilin
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Urine urobilinogen: significance
• Normally present in low amounts
• Peak levels between 2-4 pm
• Elevated in
– Liver disease
– Intestinal obstruction
– Hemolytic anemia
– Hemolysis
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Urine nitrite formation
• Nitrates normally present
• Nitrates converted to nitrite by bacteria
• Reaction takes up to 4 hours to complete
• UTIs of only non-nitrate reducers will be negative
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Urine nitrite
Nitrite color chart
Reaction A:
Nitrite + p-arsanilic acid
diazonium compound
Reaction B:
3-Hydroxyl-1,2,3,4 tetrahydrobenz-(h)-quinoline +
diazonium compound = pink color
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Urine nitrite: false positive
• Specimen left at room temperature causing bacteria to
multiple.
• Phenazopyridine
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Urine nitrite: false negative
• Specimen containing non-nitrate reducing pathogens
• Insufficient time in the bladder
• Low or no nitrate diet
• Nitrite was to nitrogen
• Elevated urobilinogen
• Ascorbic Acid
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Urine nitrite: significance
• Screening method only
• Not to replace microbiology procedures
• If there are clinical symptoms, then regular
bacteriology tests should be performed, even if the
nitrite test is negative.
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Urine leukocyte esterase
• Leukocyte esterase is present only in neutrophils.
• Few neutrophils can be seen in normal urine.
• Increased numbers of neutrophils usually indicate the
presence of a urinary tract infection.
• Screening for UTI also includes pH, protein, and nitrite.
• Mix specimen well and test at room temperature.
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Urine leukocyte esterase
Leuk. Esterase color chart
Reaction A:
Indoxyl
granulocytic
or
carbonic acid ester
pyrole
esterase
Reaction B:
indoxyl
or
+ diazonium salt = purple
pyrole
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indoxyl
or
pryole
Urine leukocyte esterase: false positive
• Strong oxidizing agents
• Contamination by vaginal discharge
• Formalin used as preservative
• Drugs containing imipenem, meropenem, or clavulanic
acid
• Nitrofurantoin contributes a color to urine that may
cause misinterpretation
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Urine leukocyte esterase: false negative
• High specific gravity and in urines containing glucose
and protein.
– WBCs creanate and cannot release esterase
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Ascorbic acid
Oxalic acid
Cephalexin
Cephalothin
Gentamicin
Tetracycline
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Additional urine chemistry parameters
• Calcium
• Creatinine
• Microalbumin
• Ascorbic acid
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Summary
• Technological advances have provided for the
development of reagent test strips (dipsticks) that
allow for the rapid simultaneous determination of
commonly measured urinary substances.
• Abnormal urine chemistry results not only aid in the
assessment of renal disorders but can also disclose
many systemic disorders.
• Adhere carefully to all manufacturer instructions to
avoid reporting of misinterpreted results.
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