KETONES IN DIABETES MELLITUS

MLS 419: AUBF LEC ➔ Ketonuria shows insulin deficiency and the need to
MIDTERMS WEEK 2 – LESSON 2 regulate dosage
CHEMICAL EXAMINATION OF URINE II ➔ Ketoacids accumulate in the patient’s plasma (decrease
in pH and bicarbonate levels)
KETONES ◆ Normal blood pH: 7.35- 7.45
➔ Intermediate products of fat metabolism ◆ Accumulation makes the pH acidic
◆ Acetone (2%)- 2nd to form ➔ Diuresis is initiated to remove glucose and ketones
◆ Acetoacetic acid (20%)- 1st to form ◆ not good without replenishment
◆ β-hydroxybutyrate (78%)- 3rd to form ➔ Chemical imbalance ensues which may lead to diabetic
➔ Present only when the use of available carbohydrates coma which may also lead to death
becomes compromised ➔ Ketones in the urine are valuable in the monitoring and
◆ Fats are metabolized to supply energy in lieu of management of type 1 DM
carbohydrates
◆ Normally no measurable ketones are produced CLASSIC TESTS FOR KETONE
● Renal threshold: 70 mg/dL Ferric chloride test
● Also eliminated in the lungs through breathing ➔ Gerhardt’s test, 1865
● Symptom of ketoacidosis: acetone breath ➔ Discontinued due to many false-positive reactions
(salicylates)
FORMATION
Nitroprusside test

➔ Legal’s test, 1883; modified by Rothera in 1908

➔ More sensitive to acetone and acetoacetate than reagent
strips
➔ Sensitivity:
◆ Acetoacetate: 1-5 mg/dL
◆ Acetone: 10-25 mg/dL
➔ Positive: Purple-colored ring formation
➔ Disadvantage: Impractical, time-consuming, laborious

REAGENT STRIP REACTIONS

➔ Normal end products of fatty acid metabolism are carbon

dioxide and water
➔ Ketogenesis
◆ happens when there is an excessive amount of fats
and occurs
◆ happens in the mitochondria of the liver
◆ limited availability of carbohydrates forces the liver
to oxidize fatty acids
➔ Most brands of reagent strips are only sensitive to ➔ Currently performed test for ketones
acetoacetate ➔ Uses sodium nitroprusside (nitroferricyanide) to
measure ketones
CLINICAL SIGNIFICANCE ➔ Acetoacetate reacts with sodium nitroprusside to
Inability to metabolize carbohydrate produce a change in color from beige to purple (large
➔ Diabetes mellitus (diabetic acidosis, insulin dosage amount)
monitoring) ◆ For Multistix
◆ Especially Type 1 ➔ The addition of glycine enables the detection of acetone
◆ Insulin- to metabolize carbohydrates ◆ For Chemstrip
Increased loss of carbohydrate ➔ Detection of ketones is qualitative (+/-) or
➔ Vomiting semi-qualitative
Inadequate intake of carbohydrate ➔ Presence of one ketone bodies is enough
➔ Starvation (several days) ➔ Sensitivity:
➔ Malabsorption/pancreatic disorders ◆ Acetoacetate: 5-10 mg/dL
◆ Acetone: 50-70 mg/dL
➔ Better test for routine urinalysis
REACTION INTERFERENCE HEMATURIA
➔ False-positive (difference: color fades rapidly on ➔ From trauma or damage of renal or genitourinary origin
standing) ◆ Renal calculi- kidney stones
◆ Phthalein dyes ◆ Glomerulonephritis- inflammation
◆ Highly-pigmented red urine (major concern) ◆ Pyelonephritis- inflammation
◆ Levodopa ◆ Tumors- swelling/inflammation
◆ Medications containing free sulfhydryl groups ◆ Exposure to chemicals and anticoagulant
(MESNA, captopril) therapy
➔ False-negative ➔ May also be due to post-strenuous exercise and
◆ Improperly preserved specimens due to rapid menstrual contamination
volatilization of ketones (remedy: test immediately
or refrigerate) HEMOGLOBINURIA
➔ Lysis of RBC produced in the urinary tract particularly in
Acetest tablets dilute, alkaline urine
◆ RBCs in microscopic exam may be seen
➔ Intravascular hemolysis
◆ No RBC in microscopic exam because hemolysis
started prior to the blood reaching the kidneys
◆ Normally: free hemoglobin forms a complex with
haptoglobin (no Hgb in urine)
◆ If all available haptoglobin is bound, free hgb is
filtered by glomerulus
◆ May be processed by renal tubular cells into
hemosiderin
◆ Hemolytic anemias, transfusion reactions,
severe burns, strenuous exercise, brown
recluse spider bites, infections (e. g. malaria)
➔ Confirmatory test for questionable ketone strip results
➔ Advantage: may be used on other specimens
➔ Contains:
◆ Nitroprusside- detect acetoacetate
◆ Glycine- detect acetone
◆ Lactose- aid in the enhancement of color reaction
(purple)
➔ 1 drop of specimen into the tablet and wait for 30
seconds
➔ Difference of concentration: intensity of the color
➔ Sensitivity:
◆ Acetoacetate: 5-10 mg/dL
◆ Acetone: 20 mg/dL (more sensitive than reagent MYOGLOBINURIA
strip) ➔ Myoglobin: heme-containing protein found in muscle
tissue
BLOOD ➔ Muscle destruction/rhabdomyolysis
➔ May be present in the urine in the form of intact red ◆ Trauma
blood cells (hematuria) or as the product of RBC ◆ Crush injuries and contact sports
destruction (hemoglobinuria) ◆ Muscle ischemia (prolonged coma, alcoholism)
➔ Most accurate method in determining the presence of ◆ Muscle infections
blood in urine ◆ Myopathy from medications
◆ 5 cells/μL of urine is clinically significant (may not ◆ Seizures/convulsions
be detected microscopically) ◆ Toxins from snake and spider bites
➔ Positive chemical test for blood is nonspecific: red blood
cells, hemoglobin, myoglobin Hemoglobinuria vs Myoglobinuria
➔ Blood reagent strip measures the presence of heme ➔ Historical test: ammonium sulfate precipitation
moiety (present in hgb and myoglobin) ➔ 2.8 g of ammonium sulfate is added to 5 mL of
centrifuged urine
CLINICAL SIGNIFICANCE ➔ Mix and let the specimen sit for 5 minutes
➔ Hematuria ➔ Filter/centrifuge urine and test supernatant with reagent
◆ intact red blood cells strip for blood
◆ cloudy or smoky red urine ➔ Hemoglobin is larger and is precipitated
➔ Hemoglobinuria ◆ Myoglobin present: supernatant remains red,
◆ Hemoglobin in urine due to RBC destruction positive for blood
◆ clear red urine ◆ Hemoglobin present: red precipitate, negative for
➔ Myoglobinuria blood
◆ tissue destruction
◆ clear red-brown urine
 Parameter Hemoglobinuria Myoglobinuria
CLINICAL SIGNIFICANCE

Urine color Pink, red, brown Pink, red, brown

Blood reagent strip test Positive Positive

Serum color Pink to red Pale yellow

(hemolysis) (normal)

Serum chemistry tests:

Haptoglobin Decreased to absent Normal
Myoglobin Normal Increased
Free hemoglobin Increased Normal
Creatinine Kinase Increased, but <10 Increased, but
times upper >10 times upper
reference limit reference limit

REAGENT STRIP REACTIONS

➔ Pseudoperoxidase activity of hemoglobin (heme
moiety)
◆ Heme component + tetramethylbenzidine
chromogen + peroxide = green-blue color

REACTION INTERFERENCE
➔ False-positive
◆ Menstrual contamination
◆ Strong oxidizing reagents
◆ Vegetable peroxidase and bacterial enzymes (e.g.
E. coli peroxidase)
➔ False-negative
◆ High SG/crenated RBCs, unmixed specimens
◆ Formalin as preservative
◆ Captopril ➔ 3 fates of urobilinogen:
◆ High concentrations of nitrite and ascorbic acid ◆ Stays in the intestine & contribute to the color of
level > 25 mg/dL stool
◆ Reabsorbed into the circulation ⇒ liver ⇒ intestine
BILIRUBIN ◆ Reabsorbed into the circulation ⇒ kidney ⇒ urine
➔ Provides an early indication of liver disease urobilinogen
Jaundice Conditions Urine Fecal ➔ 2-5% of all produced urobilinogen is sent to the kidney
Classification color via blood circulation
➔ Prehepatic Jaundice
Prehepatic Hemolytic Bilirubin: Normal
(increased disorders Negative
◆ Negative urine bilirubin
heme -Transfusion Urobilinogen: ↑ ◆ ↑urobilinogen (urine & stool)
degradation) reactions ➔ Hepatic Jaundice
-Sickle cell disease ◆ Normal/↑ urine urobilinogen
-Hereditary
spherocytosis
◆ ↑urine bilirubin (positive)
-Hemolytic disease ➔ Posthepatic
of newborn ◆ ↓urobilinogen (urine & stool)
◆ ↑urine bilirubin
Ineffective
erythropoiesis
-Thalassemia REAGENT STRIP REACTIONS
-Pernicious anemia ➔ Diazo reaction
◆ Conjugated bilirubin couples with a diazonium salt
Hepatic Hepatitis Bilirubin: Normal in an acid medium to form azobilirubin (brown in
(Hepatocellular Cirrhosis Positive
disorder) Genetic disorder Urobilinogen:
color)
Normal to ↑

Posthepatic Gallstones Bilirubin: Pale,

(Obstruction) Tumors Positive chalky,
(carcinoma) Urobilinogen: ↓ “alcoholic”
Fibrosis to absent
REACTION INTERFERENCE
➔ False-positive
◆ Highly-pigmented urine (e.g. phenazopyridine)
◆ Indican (intestinal disorders)
◆ Metabolites of Lodine
➔ False-negative
◆ Exposure to light
◆ Ascorbic acid > 25 mg/dL
◆ High nitrite concentrations
ICTOTEST TABLETS
➔ Confirmatory test for bilirubin
◆ Can detect 0.05 to 0.1 mg/dL of bilirubin
◆ Reagent strip: 0.5 mg/dL lower limit of detection
➔ 10 drops of urine to an absorbent test mat
➔ Place 1 Ictotest tablet to the moistened area
➔ Add 1 drop of water to the tablet and wait 5 seconds
➔ Add 1 drop of water to the tablet; water runs off the tablet
onto the mat
➔ Observe color change after 60 seconds (positive: from
white to blue to purple color)
UROBILINOGEN
➔ Normally present in urine in concentrations of 1 mg/dL
or less
➔ Best specimen for quantifying and monitoring: 2 hours
after mid-day meal (2-4 PM)
◆ Alkaline tide- the parietal cells, aside from
releasing hydrochloric acid, also releases
bicarbonate and induces a temporary shift from
acid to alkaline
◆ enhanced urobilinogen excretion in alkaline urine
➔ Labile in acid urine and easily photo-oxidizes into urobilin
CLINICAL SIGNIFICANCE
➔ Early detection of liver disease
➔ Liver disorders, hepatitis, cirrhosis, carcinoma
➔ Hemolytic disorders
Classic Ehrlich’s reaction
➔ Old qualitative screening for urobilinogen
➔ Urobilinogen reacts with p-dimethylaminobenzaldehyde
(Ehrlich’s reagent) in an acid medium to form a pink,
magenta, or red color
➔ 1 part Ehrlich’s reagent + 10 parts urine in a tube and
incubate for 5 minutes
➔ Nonspecific test (many Ehrlich reactive substances)
REAGENT STRIP REACTIONS
➔ Multistix: Ehrlich’s aldehyde reaction
➔ Chemstrip: diazo reaction using
4-methoxybenzene-diazonium-tetrafluoroborate (more
specific)
REACTION INTERFERENCE
➔ Multistix
◆ False-positive: Ehrlich reactive substances
◆ False-negative: old specimens, formalin
preservation
➔ Chemstrip
◆ False-positive: highly pigmented urine
◆ False-negative: old specimens, formalin
preservation, high nitrite concentrations
NITRITE
➔ Rapid screening test for the presence of UTI
➔ Valuable in detecting initial bladder infection (cystitis)
◆ Many UTIs start in the bladder and progress
upward; may also be asymptomatic
◆ Early detection of bacteriuria plus antibiotic
therapy can prevent pyelonephritis and other
complications
➔ Can be used to evaluate antibiotic therapy
➔ Can be used as periodical screen in persons at high risk
for UTI (esp. women)
REAGENT STRIP REACTIONS
➔ Based on the ability of certain bacteria to reduce nitrate
(normal constituent) to nitrite
➔ Sensitivity: 100,000 organisms/mL
➔ Greiss reaction
➔ Nitrite + aromatic amine (p-arsanilic acid or
sulfanilamide) = diazonium salt
➔ Diazonium slat + tetrahydrobenzoquinolin = pink azodye
REACTION INTERFERENCE
➔ Bacteria that lack nitrate reductase
◆ Nitrate reductase is found in many Gram-negative
bacteria that most frequently cause UTI
◆ Other organisms (non-nitrate-reducing bacteria,
yeasts, T. vaginalis) that cause UTI are not
detected
➔ Insufficient contact time between bacteria and nitrate ➔ False-negative
◆ At least 4 hours ◆ High concentrations of protein, glucose, oxalic acid,
◆ First morning specimen is ideal Vitamin C, gentamicin, cephalosporins,
➔ Lack of urinary nitrate tetracyclines
◆ Nitrate is commonly found in green vegetables ◆ Inaccurate timing (note: read LE after 2 minutes)
◆ Diet is not controlled pre-testing so false-negative
results may occur SPECIFIC GRAVITY
➔ Large quantities of bacteria further reducing nitrite to ➔ Expression of solute concentration
nitrogen ➔ A fixed SG of 1.010 regardless of hydration implies
➔ Antibiotics which inhibit bacterial metabolism significant renal tubular dysfunction
➔ Vitamin C ➔ Reagent strip measures ionic or charged solutes only
➔ High specific gravity ➔ The higher the specific gravity, the more concentrated
the urine is
LEUKOCYTE ESTERASE
➔ In conjunction with nitrite to detect the presence of UTI
➔ Normally, WBCs may be present in urine in small
numbers
◆ 20/hpf indicates pathologic process
◆ High WBC count is more often found in women than
men
➔ A more standardized test than microscopic examination
of urine sediment
➔ Not designed to measure the concentration of leukocytes
➔ Can detect lysed WBCs which are not seen in
microscopic examination
➔ Sensitivity: 10-25 WBCs/microliter
◆ Note: a negative result does not rule out increased
number of WBCs

CLINICAL SIGNIFICANCE
➔ Increased WBCs are indicators of UTI or inflammation
in the urinary tract
➔ Detects the presence of esterase in granulocytes and
monocytes (also present in Trichomonas and
histiocytes)
REAGENT STRIP REACTIONS
➔ Infections caused by Trichomonas, Chlamydia, yeast,
➔ Based on the change in pKa of a polyelectrolyte in an
and inflammation of renal tissue produce leukocyturia
alkaline medium
without bacteriuria
➔ Number of ions in a solution = ionization of
➔ Assessment of LE and nitrite tests can be cost-effective
polyelectrolyte = release of hydrogen ions
measures to determine the necessity of performing urine
➔ More concentrated urine, more hydrogen ions are
culture
released, pH lowers
REAGENT STRIP REACTIONS
Direct and indirect SG measurements
➔ Ester hydrolysis and azo coupling reaction
➔ Direct specific gravity methods determine the actual
◆ Indoxylcarbonic acid ester = indoxyl + acid
or true density of urine, regardless of the solutes
indoxyl
present. All solutes are detected and measured. Not
◆ Indoxyl + diazonium salt = purple azodye
commonly used.
◆ Examples: Urinometry, harmonic oscillation
densitometry (cause false increase)
◆ Other solutes are present because of other
abnormal processes unrelated to concentrating
ability.
◆ Impractical
➔ Indirect SG methods
◆ Reagent strip and refractometry

You are incredibly brave.

REACTION INTERFERENCE
➔ False-positive
◆ Vaginal secretions contamination
◆ Strong oxidizing agents or formalin in the container
◆ Highly –pigmented urine
◆ Nitrofurantoin