FINALS: • half-life is 20 days: glycated albumin represents

serum glucose patterns approximating 1 month.

ACTIVITY OUTLINE
• Albumin METHODS:
• Direct Bilirubin • Affinity chromatographic methods:
• Capillary puncture o based on specific interaction of boronic
acids with glycated proteins have
ALBUMIN o to determine serum concentrations of
glycated albumin.
• synthesized in the liver from 585 amino acids at
the rate of 9 to 12 g/d with no reserve or storage. PRINCIPLE COMMENT
• A protein present in highest concentration in the
plasma. Globulins are
• exists in the extravascular (interstitial) space. precipitated in high Labor
o total extravascular albumin exceeds the total salt concentrations; intensive
intravascular amount by 30% albumin in
▪ concentration of albumin: plasma Salt
supernatant is
albumin concentration = intravascular precipitation
quantitated by
albumin mass/plasma volume in the biuret reaction
blood is much greater than its
concentration in the interstitial space.
DYE BINDING:
• TRANSCAPILLARY ESCAPE RATE: Albumin leaves
the circulation at a rate of 4% to 5% of total
intravascular albumin per hour. Methyl orange Nonspecific
o measures systemic capillary efflux of
albumin. HABA Many
• Albumin is responsible for 80% of the colloid [2,4′- Albumin binds to interference
osmotic pressure (COP) of the intravascular fluid hydroxyazoben dye; causes shift in (salicylates,
o maintains the appropriate fluid balance in zenebenzoic absorption bilirubin)
the tissue acid] maximum
• Albumin buffers pH and is a negative acute-phase
BCG Sensitive;
reactant protein.
(bromocresol overestimates
• Capacity to bind various substances in the blood
green) low albumin
o 4 binding sites on albumin have varying
levels; most
specificities for different substances.
commonly
• Albumin transports thyroid hormones; other
used dye
hormones, particularly fat -soluble ones; iron; and
fatty acids. BCP Specific,
• For example, albumin binds unconjugated (bromocresol sensitive,
bilirubin, salicylic acid (aspirin), fatty acids, calcium purple) precise
(Ca2+) and magnesium (Mg2+) ions, and many Accurate;
drugs, and serum albumin levels can affect the Proteins separated gives overview
half-life of drugs. This binding characteristic is also Electrophoresis based on electric of relative
exhibited with certain dyes, providing a method charge changes in
for the quantitation of albumin. different
• clinical applications of glycated (or glycosylated) protein
albumin as a more sensitive indicator of short- fractions
term hyperglycemic control.
o Glycated hemoglobin represents trends in CLINICAL SIGNIFICANCE:
serum glucose over a period of 3 months
DECREASE:
or approximately 120 days (the life span
of red blood cells [RBCs]). • malnutrition & malabsorption
o inadequate source of amino acids
• Liver disease • Obstructive jaundice (INCREASE) alpha -
o resulting in decreased synthesis by the 2-beta globulins
hepatocytes.
o increase in globulins that occurs in early
cirrhosis, however, balances the loss in
albumin to give a total protein
concentration within acceptable limits
• Protein-losing enteropathy/ gastrointestinal loss as
interstitial fluid leaks out in inflammation and
disease of the intestinal tract as in diarrhea
• Kidney loss of albumin via urine in renal disease.
Albumin is normally excreted in very small amounts.
This excess excretion occurs when the glomerulus
no longer functions to restrict the passage of
INCREASE Dehydration
proteins from the blood as in nephrotic syndrome
• Skin loss in the absence of the skin barrier such as in NORMAL • Immunodeficiency syndromes
burns or exfoliative dermatitis • Multiple myeloma
• Hypothyroidism • Monoclonal and polyclonal
• Dilution by excess: polydipsia (drinking too much gammopathies
water) or excess administration of intravenous
fluids REFERENCE RANGE: 3.5 – 5.3 G/DL
• Acute disease states
• AUTOSOMAL RECESSIVE TRAIT: DIRECT BILIRUBIN
o analbuminemia “absence of albumin”
o bisalbuminemia “presence of albumin” • Ehrlich in 1883: “CLASSIC DIAZON REACTION”
has unusual molecular characteristics o The reaction of bilirubin with a diazotized
demonstrated by the presence of two albumin sulfanilic acid solution to form a colored
bands instead of the single band usually seen product using urine samples.
by electrophoresis. BOTH ARE RARE. • In 1913, van den Bergh: the diazo reaction may be
• Redistribution by hemodilution, increased capillary applied to serum samples but only in the presence
permeability (increased interstitial albumin), or of an accelerator (solubilizer) (ERROR)
decreased lymph clearance. • In 1937 Malloy and Evelyn: the first clinically useful
o Sepsis: a profound reduction in plasma
albumin associated with marked fluid shifts.
INCREASE: seldom clinically important
• seen only with dehydration or after excessive
albumin infusion methodology of quantitation of bilirubin in serum
samples using the classic diazo reaction with a 50%
DISEASE methanol solution as an accelerator.
• Burns • In 1938, Jendrassik and Grof: a method using the
• Trauma diazo reaction with caffeine-benzoate-acetate as
• Infections an accelerator.
• Acute (INCREASE) alpha 1 &2 globulins • PRESENT: Malloy and Evelyn: all commonly used
• Chronic (INCREASE) alpha 1 &2 gamma methods for measuring bilirubin and its fractions
globulins are modifications of the technique
• Malabsorption • Total bilirubin and conjugated bilirubin (direct
• Nephrotic syndrome bilirubin) are measured and unconjugated bilirubin
DECREASE • Salt retention syndrome (indirect bilirubin) is determined by subtracting
conjugated bilirubin from total bilirubin
Hepatic damage:
• Hepatitis (INCREASE) gamma globulins
• Cirrhosis beta & gamma bridging • BILIRUBINOMETRY in the neonatal population.
 o only useful in the neonatal population; the
presence of carotenoid compounds in adult >
causes strong positive interference in the
adult population.
o Also, involves the measurement of reflected
light from the skin using two wavelengths
that provide a numerical index based on
spectral reflectance.
o Newer generation bilirubinometers use
MICROSPECTROPHOTOMETERS
▪ determine the optical densities of
bilirubin, hemoglobin, and melanin in
the subcutaneous layers of the infant’s
skin.
▪ Mathematical isolation of hemoglobin
and melanin allows measurement of
the optical density created by bilirubin.
When using the several methods described earlier,
two of the “TOTAL BILIRUBIN: THREE FRACTIONS
OF BILIRUBIN” were identified: conjugated (direct)
and unconjugated (indirect) bilirubin.
• Unconjugated (indirect) bilirubin is a nonpolar and
water-insoluble substance; found in plasma bound
to albumin.
▪ will only react with the diazotized sulfanilic
acid solution (diazo reagent) in the presence
of an accelerator (solubilizer).
• Conjugated (direct) bilirubin is a polar and water-
soluble compound that is found in plasma in the
free state (not bound to any protein).
▪ will react with the diazotized sulfanilic acid
solution directly (without an accelerator).
• Conjugated and unconjugated bilirubin fractions
have been differentiated by solubility of the
fractions.
• Direct and indirect bilirubin results should be
reported as conjugated and unconjugated,
respectively.
• 3rd fraction of bilirubin “delta” bilirubin. A
conjugated bilirubin that is covalently bound to
albumin.
o seen only when there is significant hepatic
obstruction; molecule is attached to albumin,
it is too large to be filtered by the glomerulus
and excreted in the urine.
o when present, will react in most laboratory
methods as conjugated bilirubin.
SPECIMEN COLLECTION AND STORAGE:
• using a diazotized sulfanilic acid solution may be
performed on either serum or plasma.
o Serum: preferred for the Malloy-Evelyn
because the addition of the alcohol in the
analysis can precipitate proteins and cause
interference with the method.
• fasting sample is preferred as the presence of
lipemia will increase measured bilirubin
concentrations.
• HEMOLYZED SAMPLES SHOULD BE AVOIDED as
they may decrease the reaction of bilirubin with the
diazo reagent.
• Bilirubin is very sensitive to and is destroyed by
light; should be protected from light.
o If left unprotected from light, bilirubin
values may reduce by 30% to 50% per hour.
o If serum or plasma is separated from the
cells and stored in the dark, it is stable for 2
days at room temperature, 1 week at 4°C,
and indefinitely at −20°C.
• There is no preferred reference method or
standardization of bilirubin analysis; the American
Association for Clinical Chemistry and the National
Bureau of Standards have published a candidate
reference method for total bilirubin, “A MODIFIED
JENDRASSIK-GROF” procedure
o using caffeine-benzoate as a solubilizer; both
have acceptable precision and are adapted to
many automated instruments
• JENDRASSIK-GROF OR MALLOYEVELYN procedure
is the most frequently used method to measure
bilirubin.
JENDRASSIK-GROF METHOD:
• slightly more complex
• not affected by pH changes
• Insensitive to a 50-fold variation in protein
concentration of the sample
• Maintains optical sensitivity even at low bilirubin
concentrations
• Has minimal turbidity and a relatively constant
serum blank
• Is not affected by hemoglobin up to 750 mg/dL
Because this chapter does not allow for a detailed
description of all previously mentioned bilirubin test
methodologies, only the most widely used
principles for measuring bilirubin in the adult and
pediatric population are covered.
MALLOY-EVELYN:
• pigments in serum or plasma are reacted with a
diazo reagent.
• diazotized sulfanilic acid reacts at the central
methylene carbon of bilirubin to split the molecule
forming two molecules of azobilirubin.
• at pH 1.2: the azobilirubin produced is red-purple in ▪ Proteins in serum are eliminated with
color with a maximal absorption of 560 nm. saturated ammonium sulfate
• most commonly used accelerator to solubilize (NH4)2SO4 and then bilirubin is
unconjugated bilirubin is METHANOL, although reacted with diazotized sulfanilic acid
other chemicals have been used. forming blue azorubilirubin using
• MODIFICATION: alcoholic HCL as coupling promoter
o Thamhauser–Andersen modification Reagents and results:
▪ direct reaction is made on the serum and •
Saturated Ammonium Sulfate – protein
the indirect on the alcoholic extract precipitant
separated from serum after it has been • Alcoholic HCl – coupling promoter
precipitated by ethyl alcohol and
• Ehrlich’s diazo reagent – color reagent
ammonium sulfate.
• Blue azobilirubin at acid pH – end product and
o Anino, Watson and ducci
color
JENDRASSIK-GROF METHOD FOR TOTAL AND MODIFICATIONS: Alkaline Methanolysis Method
CONJUGATED BILIRUBIN DETERMINATION • used in researches only.
• Bilirubin pigments in serum or plasma are reacted • MOST ACCURATE METHOD for measuring bilirubin
with a diazo reagent (sulfanilic acid in hydrochloric o formation methyl esters and determination of
acid and sodium nitrite), resulting in the production the products by HIGH PERFORMANCE LIQUID
of the PURPLE PRODUCT AZOBILIRUBIN. CHROMATOGRAPHY (HPLC).
• product azobilirubin may be measured SOURCES OF ERROR:
spectrophotometrically.
• Instruments should be frequently standardized to
• The individual fractions of bilirubin are determined
maintain reliable bilirubin results
by taking two aliquots of sample and reacting one
• careful preparation of bilirubin standards is critical
aliquot with the diazo reagent only and the other
as these are subject to deterioration from exposure
aliquot with the diazo reagent and an accelerator
to light.
(caffeine-benzoate).
• HEMOLYSIS AND LIPEMIA SHOULD BE AVOIDED as
o addition of caffeinebenzoate will solubilize
they will alter bilirubin concentrations.
the water-insoluble fraction of bilirubin and
• SERIOUS LOSS OF BILIRUBIN occurs after exposure
will yield a total bilirubin value (all
to fluorescent and indirect and direct sunlight;
fractions).
o exposure of samples and standards to light
• The reaction WITHOUT THE ACCELERATOR WILL
be kept to a minimum.
YIELD CONJUGATED BILIRUBIN ONLY. After a short
• Specimens and standards should be refrigerated in
period of time, the reaction of the aliquots with the
the dark until testing can be performed.
diazo reagent is terminated by the addition of
ascorbic acid.
REFERENCE RANGE
o ascorbic acid destroys the excess diazo
reagent. The solution is then ALKALINIZED BILIRUBIN RANGE:
using an alkaline tartrate solution, which ADULT: Conjugated 0.0–0.2 mg/dL (0–3
shifts the absorbance spectrum of the bilirubin µmol/L)
“AZOBILIRUBIN” TO A MORE INTENSE BLUE Unconjugated 0.2–0.8 mg/dL (3–14
COLOR that is less subject to interfering bilirubin µmol/L)
substances in the sample. Total bilirubin 0.2–1.0 mg/dL (3–17
o final blue product is measured at 600 nm, µmol/L)
with the intensity of color produced directly
PREMATURE Total bilirubin at 1–6 mg/dL (17–103
proportional to bilirubin concentration.
INFANTS: 24 h µmol/L)
• Indirect (unconjugated) bilirubin may be calculated
by subtracting the conjugated bilirubin Total bilirubin at 6–8 mg/dL (103–137
concentration from the total bilirubin 48 h µmol/L)
concentration. Total bilirubin 3–5 10–12 mg/dL (171–
• MODIFICATIONS: days 205 µmol/L)
o Stoner and wiseberg method:
 FULL TERM Total bilirubin at 2–6 mg/dL (34–103
INFANTS: 24 h µmol/L)
Total bilirubin at 6–7 mg/dL (103–120
48 h µmol/L)
Total bilirubin 3–5 4–6 mg/dL (68–103
d µmol/L)
UROBILINOGEN IN URINE AND FECES:
• a colorless end product of bilirubin metabolism that
is oxidized by intestinal bacteria to the brown
pigment urobilin.
• normal individual, part of the urobilinogen is
EXCRETED IN FECES, and THE REMAINDER is
reabsorbed into the portal blood and returned to
the liver.
• A small portion that is not taken up by the
hepatocytes is excreted by the kidney
“UROBILINOGEN”.
CLINICAL SIGNIFICANCE:
INCREASE:
• hemolytic disease
• defective liver cell function “hepatitis”
DECREASE:
• complete biliary obstruction.
• FECAL UROBILINOGEN: biliary obstruction & HCC.
MOST QUANTITATIVE METHODS for urobilinogen are
based on a reaction first described by EHRLICH IN 1901:
• reaction of urobilinogen with p
dimethylaminobenzaldehyde (Ehrlich’s reagent) to
form a red color.
• “Determination of Urine Urobilinogen
(Semiquantitative)” most laboratories use the less
laborious, more rapid, semiquantitative method
DETERMINATION OF URINE UROBILINOGEN
(SEMIQUANTITATIVE)
• Urobilinogen reacts with p-
dimethylaminobenzaldehyde (Ehrlich’s reagent) to
form a red color, which is then measured
spectrophotometrically.
o Ascorbic acid is added as a reducing agent to
maintain urobilinogen in the reduced state.
o The use of saturated sodium acetate stops
the reaction and minimizes the combination
of other chromogens with the Ehrlich’s
reagent.
SPECIMEN:
• fresh 2-hour urine specimen
o should be kept cool and protected from light.
SOURCES OF ERROR:
• The results > in Ehrlich units rather than in
milligrams of urobilinogen because substances
other than urobilinogen account for some of the
final color development.
• COMPOUNDS, OTHER THAN UROBILINOGEN, that
may be present in the urine and react with Ehrlich’s
reagent include porphobilinogen, sulfonamides,
procaine, and 5-hydroxyindoleacetic acid. Bilirubin
will form a green color and, therefore, MUST BE
REMOVED, as previously described.
• FRESH URINE & the test must be performed
WITHOUT DELAY TO PREVENT OXIDATION of
urobilinogen to urobilin.
• spectrophotometric readings should be made
within 5 minutes after color production because
the urobilinogen aldehyde color slowly decreases
in intensity.
REFERENCE RANGE: 1 Ehrlich = 1 mg of
urobilinogen
0.1 - 1.0 Ehrlich units every 2 hours or 0.5 to 4.0 Ehrlich
units per day (0.86 to 8 mmol/d)
FECAL UROBILINOGEN
• Visual inspection of the feces is usually sufficient to
detect decreased urobilinogen.
• the semiquantitative determination of fecal
urobilinogen is available and involves the same
principle described earlier for the urine.
• carried out in an aqueous extract of fresh feces, and
any urobilin present is reduced to urobilinogen by
treatment with alkaline ferrous hydroxide before
Ehrlich’s reagent is added.
REFERENCE RANGE: 1 Ehrlich = 1 mg of
urobilinogen
75 - 275 Ehrlich units/ 100 g of FRESH FECES/ 75 to 400
Ehrlich units per 24-hour
COMPOSITION OF EHRLICH’S DIAZO REAGENT:
• Diazo A: 0.1% sulfanilic acid
• Diazo B: 0.5% sodium nitrite (NaNO2)
• Diazo blank: 1.5% hydrochloric acid (HCl)