- This document describes a laboratory experiment estimating albumin levels in serum using the dye-binding method with bromcresol green dye. - Key points: Albumin strongly binds certain dyes like bromcresol green, forming complexes with distinct absorption spectra. This allows albumin concentration measurement despite excess dye. The student estimates an albumin level of 3.915 g/dL in a serum sample, within the normal reference range of 3.5-5.0 g/dL. Interpretation of albumin levels provides information about liver function, nutrition status, and calcium levels.

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER: Prof NJAGI
HANDING DATE: 25/10/2016
SUBJECT: PRACTICAL REPPORT ON ESTIMATION OF ALBUMIN
USING DIE-BINDING METHOD:
Lando E.
©

2. INTRODUCTION
All proteins, and especially albumin, tend to react with many chemical species by means of electrostatic
and tertiary van der Waal's forces and by virtue of hydrogen bonding. Bilirubin, fatty acids, most
hormones, and many drugs are transported about the body bound to albumin. Many colored dyes in the
anionic form also possess this protein-binding property. This property has been used in attempts to devise
methods by which albumin could be measured directly, without previous precipitation of globulins.
In dye-binding methods, only those dyes or indicators can be used that bind very tightly to the albumin
molecule, so that practically 100 per cent of the albumin present is bound to dye. The binding must be
unaffected by reasonably small changes in ionic strength and pH. Further, the color of the protein-bound
dye should be different from that of the free dye; i.e., there should be a substantial shift in the wavelength
of light at which maximum absorption occurs in the two forms. The albumin-dye concentration can then
be measured in the presence of excess dye. Finally, dye binding to other protein fractions (globulins) must
be negligible if the dye binding is to be the basis for a valid assay of albumin. For practical reasons,the
color characteristics of the dye should be such that it can be measured at wavelengths of light where
bilirubin and hemoglobin will give negligible or minimal interference. The use of methyl orange was
proposed by Bracken and Klotz and Watson and Nankiville; HABA (2-(4'-hydroxyazobenzene)-benzoic
acid) by Rutstein, Ingenito, and Reynolds and Martinek, and bromcresol green by Rodkey. The use of
bromcresol green is most free of pigment interference. Bilirubin at levels over 5 mg./100 ml. interferes
with HABA procedures and to a lesser degree with methyl orange methods.
PRINCIPLES
-Many proteins have properties to bind to bind certain dyes. Expecially albumin binds specifically to
severalanionic dyes such as bromocresol green(BCG). The albumin-BCG complex absorbs maximally at
630nm. The intensity of color formed is directly proportional to albumin concentration in serum. The
interference from other proteins in serum is much minimal, because albumin binds BCGmuch faster.
BCG + Albumin PH 4.3 BCG- albumin complex
CLINICAL SIGNIFICANCE
One of the most important serum proteins produced in the liver is albumin.
This molecule has an extraordinarily wide range of functions, including nutrition, maintenance of oncotic
pressure and transport of Ca ++, bilirubin, free fatty acid, drugs and steroids .
Variation in albumin levels indicate liver diseases, malnutrition, skin lesions such as dermatitis and burns
or dehydration.
Clinical diagnosis should not be made on a single test result ; it should integrate clinical and other
laboratory data.
MATERIALS REQUIRED BUTNOT PROVIDED

3. - Spectrophotometer or colorimeter capable of measuring absorbance at 630 nm.
- Matched cuvettes 1.0 cm light path.
- General laboratory equipment.
STORAGE AND STABILITY
- Store at 2-8 °C.
- The Reagents are stable until the expiry date on the label.
-Protected the reagent from light.
- Signs ofreagent deterioration :
Presence of particales and turbidity.
Blank absorbance (A) at 630 nm ≥0.40.
REAGENT PREPARATION
The Reagent and Standard are ready to use.
SAMPLES
- Serum or EDTA plasma.
- Albumin in serum and plasma is stable for 2 weeks at 2-8 °C, and for up to 4 months at –20°C.
INTERFERENCES
- Heparin interferes with this dye binding method.
- Specimens containing dextran should be avoided.
- Lipemic samples (triglycerides > 10 g/L), require a blank correction. Use the same volume of sample
with isotonic saline in the place of the reagent.
- Hyperbilirubinemia or hemolysis does not affect the assay since the absorption maximum of the
complex absorbs at a wavelength distinct from those at which bilirubin and hemoglobin interfere.
PROCEDURE
1. Assay conditions :
Wavelength …………………..630 nm(600-650)
Cuvette …………………………1 cm light path

4. Temperature …………………15-25 oC
2. adjust the instrument to zero with distilled water.
3. Pipette into a cuvette :
TUBES Blank Sample Standard
Reagent 2.0 mL 2.0 mL 2.0 mL
Sample - 10 μL -
Standard - - 10 μL
4. Mix and incubate for 10 minutes at room temperature (15-250C).
5. Read the absorbance (A) of the samples and standard, against the blank.
The color is stable for 60 minutes at room temperature.
OBSERVATIONS AND RSELTS
Sample Standard Blank
R(µl) 2.0 2.0 2.0
standard(µl) - 10 -
sample(µl) 10 - -
Absorbance(O.) 0.505 0.645 0.090
Calculation:
Concentration of albumin in serum (g/dL) =
= (0.505 / 0.645) x 5
=3.915g/dl
=3.915mg/dl x 144.9
=567.28mol/L
The concentration of Albumin in these serum is of normal level.
Conversion factor: g/dl X144.9 = μmol/L
REFERENCE VALUES
3.5 to 5.0 g/dl
Reading of Test x 5
Reading of Standard

5. DISCUSIONS :
Clinical uses ofmeasurements and interpretation ofresults
Liver function :Although albumin is synthesised in the liver, confounding factors limit the value of single
measurements as an index of liver function. Its relatively long half life may cause its concentration to rem
ain normal in the early stages of even severe acute liver disease. A falling concentration in chronic liver di
sease suggests a clinically significant deterioration in liver function ‘decompensation’).
2. Nutrition :Although widely regarded as a ‘nutritional protein’, [albumin] is a poor guide to nutritional s
tatus. In simple starvation, the catabolic rate of albumin falls, and this and contraction of ECF volume
may cause its concentration to remain normal. Low concentrations, except in severely starved patients, su
ggest increased catabolism (e.g. due to sepsis) or increased loss (e.g. due to protein‐losing enteropathy)
3. Interpretation of [calcium] Because approximately 50% of calcium is bound to albumin in the blood,
total calcium concentration depends in part on [albumin]. Most analysers employ a formula to ‘adjust’ me
asured [calcium] for abnormal [albumin] e.g. ‘adjusted’ calcium = 0.02(40 [albumin]) + [measured calciu
m] (units: albumin g/L; calcium mmol/L).
Confounding factors
The many factors that can affect [albumin] make it essential that all are considered before ascribing an ab
normal value to any one. Sick patients may have low [albumin] for a combination or reasons, e.g. sepsis,
protein loss, decreased synthesis and fluid imbalance.
Causes and investigation ofabnormal values
High concentrations:High concentrations are unusual. The only causes are:
• water depletion • recent infusion of plasma or other albumin‐containing fluids.
7.2 Low concentrations
Causes of Low concentrations are common.
The causes are: • decreased synthesis or inadequate nitrogen intake, malabsorption ,
chronic liver disease, increased catabolism, sepsis, other catabolic states, increased plasma volume,
water excess • redistribution( ascites, oedema, sepsis)• increased loss( protein‐losing enteropathy,
nephrotic syndrome, loss of plasma, e.g. from burns).
CONCLUSION:
Normal globulins and most abnormal globulins have no effect on the albumin values. Occasionally
paraproteins and macroglobulins will interfere by giving high results. Dye binding, being a dissociation-
association equilibrium phenomenon, is influenced by changes in temperature; a rise in temperature
increases the dye-albumin dissociation and thus gives lower results. Therefore,standards and sera should
be run under identical conditions. Heparinized plasma should not be used because heparin, for reasons not
understood, enhances dye binding by albumin. Serum albumin levels obtained by methyl orange dye

6. binding techniques tend to be 0.1 to 0.2 gm./100 ml. higher than those obtained by salt-fractionation or
electrophoresis, although some chemists have reported results as much as 0.5 gm./100 ml. higher.
there is considerable disagreement over the reliability of the dye-binding methods. Occasionally, albumin
values obtained are unusually high as a result of the binding of the dye to proteins other than albumin
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Doumas, B. T.; Bayse,D. D.; Carter,R. J.; Peters Jr,T. and Schaffer,R. (1981), A candidate reference
method for determination of total protein in serum. I. Development and validation. Clin. Chem.,27,1642-
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Feigl, F. (1975), Spot test in organic analysis.7th
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Goren, M. P. and Li, J. T. L (1986), The Coomassie brilliant blue method underestimates drug-induced
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Gornall, A. G.; Bardawill, C. J. and David, M. M. (1949), Determination of serum proteins by means of
the biureto reaction. J. Biol. Chem.,177,751-766.