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Detoxicol

SDLS 2008
Medicine for the intoxicated
File Created on 12/13/2004 05:08 PM Topic: Experiment 5Serum Total Protein and Albumin Determination
Transcriber(s): Pre Ausan and JC Tayco Lecturer: Dr. Ma. Esperanza E. Uy and Prof. Patricia Generoso
Editors: JC Tayco, Mark Lomboy, Pre Ausan, Erika Mendoza No. of pages: 2

EXPERIMENT 5: SERUM TOTAL PROTEIN AND A/G RATIO


ALBUMIN DETERMINATION ♣ Total Protein = Albumin + Globulin
♣ Normal A/G ratio : 2:1
Procedure: ♣ Albumin has a higher concentration than globulin
♣ Pipette 5.5 ml of 23% Na2SO4 solution to 0.5 ml serum, ♣ The A/G ratio signifies certain abnormalities in serum
cover with parafilm and mix by inversion (3times) protein composition. It also includes the presence of
♣ Remove 2 ml of the solution for total protein albumin in a disease, or elevations of one or more
determination globulin fraction.
♣ Remaining 4 ml sol’n + 2 ml diethylether. Cover with ♣ A decrease in the A/G ratio from normal shows there is
parafilm and mix (by inversion) for 10 seconds. an increase in the globulin concentration in
Release pressure each time by opening the cover to comparison to albumin concentration
prevent the spill of contents ♣ The plasma of patients with liver disease often shows
♣ Centrifuge 2 minutes. Two layers will be formed a decrease in A/G ratio.
♣ Pipet 2 ml solution from the bottom (albumin layer) and ♣ Synthesis of albumin decreases relatively early in
reserve for the Biuret Test. conditions of protein malnutrition, such as
♣ Pipet 2.5 ml Biuret reagent to each tube. Mix and place kwashiorkor
in a 37OC water bath for 5 minutes.
♣ Cool and read at 540 nm wavelength Background
♣ Calculate the amount of proteins in grams/dL using the ♣ Plasma-fluid matrix composing of 55% of blood
following formula: volume; contains many proteins w/c are synthesized
Reading of Unk x Conc.of Std x Dil factor = in the liver and are glycosylated
g/dL protein ♣ Serum-liquid portion which does not have an intact
Reading of Std clotting and fibrinogen
♣ Plasma proteins:
♣ Additional notes: o >200, most are glycoproteins
o Na2SO4 – used to extract the protein o 3 major groups of plasma proteins
o Diethylether – used to extract the albumin  Albumin: 4 – 5 g/100 m
fraction (lower layer)  γ – Globulin: 1g/100
o Middle Portion – globulin and other non-  Fibrinogen: 0.2 – 0.14 g/100 ml
protein components (lipids)
o Upper layer – ethyl portion ALBUMIN
♣ Major protein in human plasma
Sample Computation: ♣ Is not glycosylated
♣ Serum Total Protein: ♣ Principal determinant of intravascular osmotic pressure
0.742 x 2mg x 6 x 1 = 37.728 mg/ml ♣ Binds with many ligands such as drugs and bilirubin
0.236 2 0.5 ml ♣ MW 69,000
♣ Single Chain, 585 amino acids, 50% α helix
37.728 mg x g x 100 ml = ♣ Shape is ellipsoidal causing only slight increase in
3.7728 g/dL viscosity
ml 1000 mg 1 dL ♣ 40% intravascular, 60% located in interstitial space
♣ Synthesized in liver
*Same formula is used for the computation of albumin ♣ Liver failure may result in hypoalbuminemia→ EDEMA
fraction.
GLOBULIN
♣ Normal Values: ♣ Another plasma protein
o Total Protein: 6-8 g/dL ; 7.0 – 7.5 g/dL ; ♣ If levels are altered – may indicate liver diseases
60 – 80 g/L ♣ Increase: indicates chronic hepatitis
o Albumin : 3.5 – 5.5 g/dL ; 3.4 – 4.7 g/dL ; ♣ Decrease: indicates chronic liver disease
35-55 g/L
o Globulin : 2.3-3.5 g/dL Increased Serum Protein – indicative of:
♣ Rheumatoid Arthritis
TOTAL PROTEIN DETERMINATION ♣ Collagen disease
♣ Acute liver disease
♣ Cupric ions (Cu2+) complex with the groups involved in ♣ Chronic infection
the peptide bond in an alkaline medium and in the
♣ Multiple Myeloma
presence of at least 2 peptide bonds, a violet colored
chelate is formed
Decreased Serum Protein – indicative of:
♣ Absorbance of the colored chelate is measured at 540
♣ Increase loss of albumin in urine due to nephritic
nm
disease
♣ The color that is formed is proportional to the number
♣ Low protein intake such as in kwashiorkor
of the peptide bonds in this protein
♣ Decreased formation in the liver
♣ Diarrhea
♣ Severe hemorrhage
♣ Severe burns
♣ Exfoliative dermatitis
♣ Malabsorption

Increased Serum Albumin – indicative of:


♣ Dehydration
♣ Intravenous Albumin inclusion

Decreased Serum Albumin – indicative of:


♣ Diarrhea
♣ Starvation
♣ Impaired synthesis (liver disease, chronic infection)
♣ Increased breakdown (trauma)
♣ Increased loss (edema, burns, hemorrhage, nephritic
syndrome, protein-losing enteropathy)
♣ Congenital deficiency
♣ Hypocalcemia

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