LABORATORY METHOD (TOPIC 2 PROTEINS) Flashcards
Total Protein preferred Specimen
Serum, non-fasting
Interferences of Total protein specimen consideration
Lipemia
Hemolysis
Low total protein at?
Birth
During pregnancy
Elderly
Concentration is slightly low in recumbent position than ambulatory due to shift in water distribution.
Total protein
Specimen that can also be used for Total Protein Determination
Urine 24H
CSF
Total protein determination Method
Kjeldahl method
Biuret reaction
Lowry method
Dye binding by BRADFORD
Refractometry
Salt precipitation Test
UV absorption
Serum Electrophoresis
What TOTAL PROTEIN METHOD
Measure nitrogen content after protein digestion.
Boric ammonia is TITRATED with HCL to determine the amount of nitrogen present in the solution.
Precise and accurate but time consuming.
Kjeldahl method
Serum protein is treated with CuSO4 in alkaline medium forming purple/violet color.
color intensity is directly proportional to the concentration of protein in solution.
BIURET REACTION (colorimetric)
Most widely used method for TP determination.
BIURET REACTION (colorimetric)
Buret Reagent for Biuret Reaction
CuSO4
NaOh
Rochelle Salt (potassium-sodium-tartate)
Kl
Biuret Reaction Modifications: Kingsley and Weischelbaum – uses _____
22-26% Na2SO4
Lowry Method aka
Folin Ciocalteau
based on the reaction of TRYPTOPHAN and TYROSINE residues of protein with Folin ciocalteau amixture of PHOSPHOTUNGTIC ACID and PHOSPOMOLYBDIC ACID producing BLUE colored complex.
LOWRY METHOD (Folin ciocalteau)
dye binds amine group of amino acid residues in polypeptide chain
DYE BINDING by BRADFORD
Dyes used in DYE BINDING by BRADFORD
Bromcresol purple(BCP)- the most sensitive
Bromcresol green (BCG)- the commonly used
Tetrabromphenol blue- used in urine reagent strip. Specific to albumin
Ninhydrin- specific for amino acids
Pyrogallol red- for urine and CSF protein
the most sensitive DYE BINDING by BRADFORD
Bromcresol purple(BCP)-
the commonly used DYE BINDING by BRADFORD
Bromcresol green (BCG)
used in urine reagent strip. Specific to albumin by DYE BINDING by BRADFORD
Tetrabromphenol blue-
specific for amino acids by DYE BINDING by BRADFORD
Ninhydrin
for urine and CSF protein by DYE BINDING by BRADFORD
Pyrogallol red-
Rapid estimation of protein thru measurement of refractive index.
Used in urine protein determination.
REFRACTOMETRY
SALT PRECIPITATION TEST
For urine and CSF protein
- precipitates all protein
- precipitates all globulin
Sulfosalicylic acid test (SSA)-
Trichloroacetic acid test (TCA)-
based on the protein’s ability to absorb UV light at 280 nm
UV ABSORPTION
Normal value of Total Protein
6.5-8.5 g/dl
To convert in g/L multiply by 10
most important plasma proteins with low molecular weight compared with most of the other proteins.
Albumin
Maintain colloid osmotic pressure of plasma.
Transport of amino acids from liver to tissues.
Serves as transport protein or carrier protein.
Albumin
Most widely used laboratory method for Albumin
Dye binding methods
Albumin Normal Value
3.5-5.5 g/dl
Clinical significance absence of albumin
Analbuminemia
presence of 2 bands in the albumin region (electrophoresis)
Bisalbuminemia
decrease level of albumin
Hypoalbuminemia
Clnical Sinificance Albumin increase in?
Shock
Acute dehydration
Multiple Sclerosis
Hypothyroidism
Addisons disease
Multiple myeloma
Diarrhea
Clnical Sinificance Abumin Decrease in?
Malnutrition and Mal-absorption
Liver disease
Nephrotic syndrome
Burns
Extensive bleeding
Edema
Last two trimester of pregnancy
Group of 20 or more proteins grouped on the basis of solubility
Globulins
Globulins consists of?
Alpha and Beta globulins
Gamma globulins
Globulin synthesized in the liver
Alpha and Beta globulin
Globulin synthesized in the plasma cells
Gamma globulin
Globulins determination
separate ALBUMIN and GLOBULIN
globulins precipitate in 27% NaSO4 while albumin stays in solution
DIFFERENTIAL SOLUBILITY OF SALT SOLUTION
Globulins determination
direct colorimetry
TRYPTOPHAN residue reacts with GLYOXYLIC ACID in the presence of STRONG ACID MEDIUM producing purple color and read at 555 nm
HOPKINS-COLE METHOD
Globulins determination
Determine the concentration of TOTAL PROTEIN and ALBUMIN GLOBULIN is determined indirectly by difference.
BIURET METHOD
Globulin Biuret method Normal Value
2.0-3.5 g/dl
Globulin Formula
Total protein - albumin
use to measures the total amount of plasma proteins
A/G RATIO (ALBUMIN/GLOBULIN)
A/G RATIO (ALBUMIN/GLOBULIN) normal value
1.1-1.8:1
A/G ratio increase in?
Dehydration
High protein diet
Corticosteroid
Growth Hormone
A/G ratio decrease in?
Liver disease
Infectious diseases
Multiple myeloma
Nephrotic syndrome
