RBC Protoporphyrins Flashcards Preview

Clinical Chemistry Laboratory 2 > RBC Protoporphyrins > Flashcards

Flashcards in RBC Protoporphyrins Deck (26)
Loading flashcards...
0

EP levels due to lead toxicity

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

1

Normal value of protoporphyrin

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

2

Calculation

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

3

Grade of all chemicals

Reagent grade

4

Reagents

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

5

Extracted in 1.5 N HCl

Free erythrocyte protoporphyrin

6

Procedure

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

6

Extracts the protoporphyrins

Formic acid:diethyl ether mixture

8

Specimen

Whole blood

9

Principle

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.

10

Functions of acetone:ethyl acetate mixture

1 Lyses the red blood cells
2 Frees the protoporphyrin from other organic substances

11

Porphyrins of clinical significance

1 Uroporphyrins
2 Coproporphyrins
3 Protoporphyrins

12

Screening tests for porphyrins or their precursors

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

13

What are porphyrins?

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

14

Basis of appropriate quantitative measurements

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

15

Function of Soret band

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

16

T or F. Few porphyrins are found in nature

T

17

EP levels associated with iron deficiency

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

18

Lead toxicity manifestation

Increased erythrocyte protoporphyrin

19

Wavelengths

380 nm
405 nm
430 nm

19

Soret band

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

20

Disorders with increased erythrocyte protoporphyrin

Iron deficiency anemia
Eythropoietic porphyrias

21

Normal value of erythrocyte coproporphyrin

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

23

General procedure

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

24

Exhibited by all porphyrins

Characteristic red fluorescence

25

What are porphyrias?

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