Amino Acid and Protein Analytical Methods

Question 1

List the reference ranges for total protein and albumin, including the correct reporting units.

Answer 1

a. total protein: 6.8-8.3 g/dL

b. albumin: 3.4 – 5.0 g/dL

Question 2

Contrast the principles and reagents used in the following analytical techniques for protein measurement:

Kjeldahl Method

Answer 2

a. Based on quantitation of nitrogen content of protein (multiply by 6.25 to obtain value)

Question 3

Contrast the principles and reagents used in the following analytical techniques for protein measurement:

Biuret Method

Answer 3

a. Biuret reagent, in the presence of copper, yields a violent color – the intensity of the color is proportional to the number of peptide bonds

Question 4

Contrast the principles and reagents used in the following analytical techniques for protein measurement:

Refractometry

Answer 4

a. Based on the refractive index of light – the ratio of light refraction in the two differing media is related to protein concentration

Question 5

Contrast the principles and reagents used in the following analytical techniques for albumin measurement:

Dye-binding

Answer 5

a. Dye binding: using bromcresol green/purple, the color produced by albumin changes the absorption wavelength from the dye wavelength

Question 6

Define “electrophoresis”

Answer 6

a. The migration of charged solutes/proteins in a liquid medium under the influence of an applied external electrical field

Question 7

List three typical support media for electrophoresis

Answer 7

1. Agarose gel
2. Cellulose acetate
3. Polyacrylamide gel

Question 8

Name the charge at the anode and the charge at the cathode in an electrophoretic chamber

Answer 8

a. Anode: positive
Cathode: negative

Question 9

Explain the indications for performing immunofixation electrophoresis

Answer 9

a. Identifies monoclonal or polyclonal gammopathies

Question 9

List the specific protein that is detected by a urine dipstick.

Answer 9

a. Albumin

Question 10

List three quantitative methods for urine protein.

Answer 10

a. Folin-Ciocalteu Method
Turbidmetry
Nephelometry

Question 11

List four methods to quantitate CSF protein.

Answer 11

1. Folin-Ciocalteu reagent reacting with tyrosine and tryptophan residues: adapted in Lowry method
2. UV absorption of tyrosine and tryptophan residues
3. Precipitation
4. Dye-binding methods

Question 11

State the two general reasons for increases in cerebrospinal fluid (CSF) protein.

Answer 11

1. Increased endogenous production (i.e. multiple sclerosis)
2. Damage to the blood-brain barrier

Question 12

Briefly explain how the IgG index is used to evaluate patients with either increased endogenous protein production (as in multiple sclerosis) vs. patients with a compromised blood-brain barrier.

Answer 12

Based on the formula, with IgG (CSF) being in the numerator, with a patient suspected of MS, there CSF index would be higher. If the Brain-Blood barrier has been compromised, more albumin would be in the CSF. This number is in the dominator of the formula; as a result, the CSF index would be lower