RBC Protoporphyrins

Question 0

Normal value of protoporphyrin

Answer 0

4.0-52.0 mcg/dL (7.2-93.6 nmol/L)

Question 1

EP levels due to lead toxicity

Answer 1

Markedly elevated: >/= 300 mcg/dL (>/= 5.4 mmol/L)

Question 2

Calculation

Answer 2

mcg/100 mL RBC = {[2(A405) - (A380 + A430)] x 1.28 x mL HCl x 100}/Hematocrit x mL of whole blood

Question 3

Grade of all chemicals

Answer 3

Reagent grade

Question 4

Reagents

Answer 4

1 Acetone
2 Ethyl acetate
3 Formic acid (98-100%)
4 Diethyl ether
5 1.5 N HCl
6 Solvent mixtures
a. Acetone: Ethyl acetate mixture
b. Formic acid: Diethyl ether mixture

Question 5

Extracted in 1.5 N HCl

Answer 5

Free erythrocyte protoporphyrin

Question 6

Procedure

Answer 6

1 Pipette 2 mL of whole blood into a 15 mL test tube
2 Add 2 mL of acetone:ethyl mixture and stir the contents of the tube vigorously with a glass rod for about 1 minute
3 Add 4 mL of formic acid:ether mixture and again stir the contents of the tube vigorously for 1 minute
4 Centrifuge the tube at full speed for about 4 minutes in order for the precipitated protein to be solidly packed
5 Decant the supernatant fluid into 15 mL graduated centrifuge tube. To the remaining precipitate, add a second 4 mL quantity of formic acid:ether mixture. Centrifuge and collect the supernatant. Combine this supernatant fluid with that from the first extraction
6 Add 2 mL of 1.5 N HCl to the combined supernatant fluid
7 Stopper the tube closely with a friction-fitting stopper and then shake vigorously for about 30 seconds. Record the volume of the lower layer of HCl
8 Transfer the HCl layer to absorbance of solution against a 1:5 N HCl blank at 380, 407, and 430 nm

Question 6

Extracts the protoporphyrins

Answer 6

Formic acid:diethyl ether mixture

Question 8

Specimen

Answer 8

Whole blood

Question 9

Principle

Answer 9

Whole blood is mixed with acetone:ethyl acetate mixture to lyse the red blood cells and free the protoporphyrin from other organic substances. Formic acid:diethyl ether mixture is then added to extract the protoporphyrins. The free erythrocyte protoporphyrin is finally extracted in 1.5 N HCl and read spectrophotometrically at 380, 405, and 430 nm.

Question 10

Functions of acetone:ethyl acetate mixture

Answer 10

1 Lyses the red blood cells

2 Frees the protoporphyrin from other organic substances

Question 11

Porphyrins of clinical significance

Answer 11

1 Uroporphyrins
2 Coproporphyrins
3 Protoporphyrins

Question 12

Screening tests for porphyrins or their precursors

Answer 12

First step in the complete laboratory investigation of any disorder of porphyrin metabolism

Question 13

What are porphyrins?

Answer 13

Metabolic intermediates in the biosynthetic pathway with heme as their principal product

Question 14

Basis of appropriate quantitative measurements

Answer 14

Preliminary extraction and differentiation by solvent partition followed by spectrophotometric or fluorometric measurements

Question 15

Function of Soret band

Answer 15

Serves as the basis for the screening test of porphyrins in biological specimens

Question 16

T or F. Few porphyrins are found in nature

Answer 16

T

Question 17

EP levels associated with iron deficiency

Answer 17

50-249 mcg/dL (0.9-4.48 mmol/L)

Question 18

Lead toxicity manifestation

Answer 18

Increased erythrocyte protoporphyrin

Question 19

Wavelengths

Answer 19

380 nm
405 nm
430 nm

Question 19

Soret band

Answer 19

Characteristic red fluorescence exhibited by all porphyrins when irradiated with light at a wavelength near 400 nm

Question 20

Disorders with increased erythrocyte protoporphyrin

Answer 20

Iron deficiency anemia

Eythropoietic porphyrias

Question 21

Normal value of erythrocyte coproporphyrin

Answer 21

0.5-2.0 mcg/dL (0.75-3.00 nmol/L)

Question 23

General procedure

Answer 23

Extraction of porphyrins into an organic solvent system (e.g. Acetic acid/ethyl acetate), followed by re-extraction into HCl

Question 24

Exhibited by all porphyrins

Answer 24

Characteristic red fluorescence

Question 25

What are porphyrias?

Answer 25

A group of inherited and acquired disorder characterized by aberrations in the activities of specific enzymes of the heme biosynthetic pathway