To measure the levels of methaemoglobin
To measure the levels of methaemoglobin.
INTRODUCTION
Methemoglobin is a form of hemoglobin that has been oxidized, changing its heme iron configuration from the ferrous (Fe2+) to the ferric (Fe3+) state. Unlike normal hemoglobin, methemoglobin does not bind oxygen and as a result cannot deliver oxygen to the tissues. Causes of methemoglobinemia may be congenital or acquired (due to various drugs or toxins).
Principle:
When the Fe++ of the heme is oxidized to Fe+++, methemoglobin is formed. Normally the auto-oxidation of ferroheme is prevented by its position in the hydrophobic heme pockets. Small quantities of methemoglobin formed in the erythrocytes are promptly reduced back to the ferrous state by enzyme systems. Methemoglobin is capable of combining with negatively changed ions such as chloride to form hematin chloride and with cyanide ions to form cyanmethemoglobin. Normally the methemoglobin level is kept low in red blood cells by the effective reductase system involving NADPH. Normal blood has less than 1% of methemoglobin. The normal absorption spectrum of oxyhemoglobin has very little optical density above 600 nm. The absorption spectrum of methemoglobin exhibits a small, characteristic peak at 630 nm. This peak is abolished as methemoglobin is converted to cyanmethemoglobin upon addition of potassium cyanide, and the drop in optical density is proportional to methemoglobin concentration.
SPECIMEN TYPE
Whole Blood EDTA
NORMAL RANGE
A normal methemoglobin fraction is about 1% (range, 0-3%).
REQUIREMENT
Equipment
Analytical instrument
spectrophotometer.
Reagents
Potassium ferricyanide, 4 % (w/v) in distilled water. It should be stored in a dark bottle, and be prepared monthly.
• Phosphate buffer solution, 0.1 M, pH 6.8.
• Phosphate buffer solution, 40 mM, pH 6.9. It can be prepared by dilution of two parts of the above reagent with three parts of distilled water.
• Potassium cyanide, 5 % (w/v) dissolved in the above 40 mM phosphate buffer solution. It should be prepared just before use.
PROCEDURE
1. A 6-mL volume of distilled water is placed in a test tube. A 0.2-mL volume of whole blood is added to the tube and mixed well.
2. After allowing the tube to stand for 5 min, 4-mL of 0.1 M phosphate buffer solution is added to the mixture and mixed well.
3. The hemolysate is centrifuged at 3,000 rpm for 10 min, and the clear supernatant is transferred to another test tube. The pH of the supernatant should be around 6.9.
4. Four 4-mL volume cuvettes of the same type are cleaned well by washing with distilled water. Th e four cuvettes are designated as A, B, C and D.
5. To cuvettes A and B, a 0.5-mL volume each of distilled water is added. To cuvettes C and D, a 0.5-mL volume each of 4 % potassium ferricyanide solution is added.
6. To cuvettes A and C, a 3-mL volume each of 40 mM phosphate buffer solution is added. To cuvettes B and D, a 3-mL volume each of the supernatant of the above hemolysate is added. Each cuvette is mixed well.
7. The absorbance of cuvette B at 630 nm is read using cuvette A as reference, and this reading is A1. After allowing cuvette D to stand for 10 min, the absorbance at 630 nm is read using cuvette C as reference, and this reading is A3.
8. To all cuvettes, a 30-µL volume each of 5 % potassium cyanide solution is added and mixed well.
9. After allowing the cuvettes to stand for 2 min, the absorbances of cuvettes B and D at 630 nm are read using cuvettes A and C as references, respectively. These readings are A2 and A4, respectively.
10. The percentage of Met-Hb is calculated by the following equation: Met-Hb % = 100 (A1–A2)/ (A3–A4).
RESULT
The methemoglobin fraction of the given sample is 1.5%.
OBSERVATION
A normal methemoglobin fraction is about 1% (range, 0-3%).
Symptoms associated with higher levels of methemoglobin are as follows:
1. < 10% - None (patients with underlying diseases may have more symptoms at lower level).
2. 10-20% - Slight discoloration (eg, pale, gray, blue) of the skin.
3. The physiologic level of methemoglobin in the blood is 0% to 2%.
4. Methemoglobin concentrations of 10% to 20% are tolerated well, but levels above this are often associated with symptoms. Levels above 70% may cause death.
5. Symptoms also depend on the rapidity of its formation.
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this content is only for your knowledge.
- Methemoglobin is a form of hemoglobin that is less able to bind to oxygen1
- Methemoglobinemia is a condition where there are elevated levels of methemoglobin in the blood, which impairs oxygen delivery to tissues2
- The administration of many drugs commonly used in hospitals–such as lidocaine, benzocaine, dapsone, and nitrates–may result in acquired methemoglobinemia
To diagnose methemoglobinemia, your doctor may order tests like:
- complete blood count (CBC)
- tests to check enzymes
- examination of blood color
- blood levels of nitrites or other drugs
- pulse oximetry to check the saturation of oxygen in your blood
- DNA sequencing
Clinical Significance
Methemoglobin, Blood - The iron in Hemoglobin is normally maintained in the ferrous state by several effective mechanisms so that only a small percentage of the total hemoglobin is in the form of Methemoglobin. The production of Methemoglobin by oxidation of the iron to the ferric state, renders the hemoglobin incapable of interacting reversibly with molecular oxygen. The oxygen affinity of the unaffected heme is also increased, resulting in the decreased release of oxygen to the tissues at low partial pressure of oxygen.
Methemoglobinemia is usually caused by exposure to various chemical agents, many which contain nitrogen. An increase in Methemoglobin can also occur due to Methemoglobin Reductase Deficiency (Congenital Methemoglobinemia).
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https://www.healthline.com/health/methemoglobinemia
https://www.healthline.com/health/normal-blood-oxygen-level
https://www.slideshare.net/AlHusseinRagab/methemoglobinaemia
https://www.slideshare.net/smcmedicinedept/two-cases-of-methemoglobinemia
https://www.slideshare.net/mhnsathish/hemoglobin-synthesis-93914017
https://www.slideshare.net/nileshkate79/haemoglobin-58248275
https://www.medicalnewstoday.com/articles/319457
https://www.healthline.com/health/methemoglobinemia
https://medlineplus.gov/ency/article/000562.htm
https://slideplayer.com/slide/17588660/
https://www.osmosis.org/notes/Methemoglobinemia
https://www.uptodate.com/contents/image/print?imageKey=HEME%2F127511
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