SPECTROMETRIC MEASUREMENT OF METHEMOGLOBIN WITHOUT INTERFERENCE OF CHEMICAL OR ENZYMATIC REAGENTS

Prof. Dr. Paulo Cesar Naoum

Janaína Radispiel

Magaly da Silva Moraes

Abstract
The authors introduce a new technique to measure the methemoglobin in relationship to oxyhemoglobin, without interference of chemical reagents or enzymes. The methodical basis sets out to detect real values of methemoglobin in the blood. To determine the level of methemoglobin in relationship of oxyhemoglobin, the blood was hemolysed and the hemoglobins (metha and oxy) were stabilized in a phosphate buffer M/60 (or 60 mol L-1 ) pH 6.8. The levels of methemoglobin and oxyhemoglobin were obtained by spectrophotometric absorption at 630nm and 540nm. This technique was compared with the technique of Evelyn and Malloy, whose methodology measures the heme-structural subproducts of hemoglobin treated with potassium ferricyanide and cyanide solutions. This technique has the advantage of removing the interference of chemical reagents or enzymes without toxic risks. The values obtained with this standardization showed that the normal levels range between 1.9% and 3.8%. Rev. bras. Hematol. Hemoter. 2004; 26 (1):19-22.



Introduction
Methemoglobin may arise from three different causes: (a) excessive formation of this pigment; (b) diminished reconvertion of methemoglobin to oxyhemoglobin; (c) abnormality molecular on histidine that bind to the prosthetic group. The first two causes result in a normal methemoglobin with increased concentration, but the third cause results an abnormal type of methemoglobin (Hb M).

Many chemicals are capable of causing toxic methemoglobinemia, usually a nitro or amino derivative of benzene. The mechanism whereby these chemicals are able to produce excessive ferrihemoglobin that supplant the activities of antioxidant enzymes, specially the methemoglobin reductase. So, the toxic methemoglobinemias is an indication of oxidative stress within the red cell.


The diminished reconvertion of methemoglobin to oxyhemoglobin depends on enzymes present in the red cells called diaphorase or NADH-linked methemoglobin reductase. The majority of methemoglobinemias are caused by a malfunction of diaphorase. This enzyme deficiency is inherited as a Mendelian recessive condition.


For last, the chemical abnormality of the hemoglobin M are due to three situation: substitutions of the distal histidyl on the alpha globin that result in the Hb M Boston (alpha 58 His to Tyr) and Hb M Iwate (alpha 87 His to Tyr); substitutions of the proximal histidil on the beta globin that result in the Hb M Saskatoon (beta 63 His to Tyr), Hb M Zurich (beta 63 His to Arg) and Hb M Hyde Park (beta 92 His to Tyr); and hemoglobin M due to a substitution in the region of the hem: Hb M Milwaaukee (beta 67 Val to Glu).


All pigments of hemoglobin (oxyhemoglobin, methemoglobin and carboxyhemoglobin) are of clinical importance, and each has a characteristic absorption spectrum demonstrable by simple spectroscopy or, more definitly, by spectrophotometry. There are some methods to measure the methemoglobin with interference of chemical reagents or enzymes. In this paper we introduce a new technique to measure the methemoglobin by spectrophotometric absortion at 630nm (methemoglobin) and 540nm (oxyhemoglobin) in hemolysed red cells stabilized in a phosphate buffer M/60.




Methods to spectrometric determination

Reagents
Saponin 1%
Phosphate buffer M/60 pH 6.8
	Na2HPO4.122O _________ 2,25g
	KH2PO4 _______________ 1,42g
	Destilled H2O q.s.p. ______1 L

 

Principle:
Methemoglobin has a maximum absortion at 630nm and oxyhemoglobin has absortion at 540nm. In the red cells both pigments coexist and they can be measured in their respectives absortions.

Methods:
In a tube A mix 100ul of whole blood with 100ul of saponin 1%, homogenize to make hemolyse and add 6ml of Phosphate buffer M/60 pH 6.8. In a tube B with 3ml of Phosphate buffer M/60 pH 6.8 add 300ul of solution from tube A and homogenize. Measure the absorbance of tube A in a spectrophotometer at 630nm, and the tube B at 540 nm. All the measurements are made against a blank containing phosphate buffer M/60 pH 6.8.

Calculation:

% Meta Hb =	[A] Tube A x 100
	[A] Tube A + [A] Tube B x 10

Results:
We compare the present method with cyanid/ferricyanid method of Evelyn and Malloy on 30 blood sample from healthy people (age: 12 to 73 years old) of both sex. The results can be appreciate on the table 1.


Table 1: Values obtained of the analyses from 30 blood samples by spectrophotometric (Meta Hb) and cyanide/ferricyanide analyses (CN-Meta Hb).

	Meta Hb (%)	CN – Meta Hb (%)
X	2,90	0,78
SD	0,49	0,38
1 SD	2,41 – 3,39	0,40 – 1,16
2 SD	1,92 – 3,88	0,02 – 1,54

The coefficient of linear correlation to determine the reprodutibility and sensitivity techniques between both methods was near 1 (r = 0,903) and this suggest positive linear correlation.



REFERENCES:

1. Lehmann H, Huntsman RG. Man's Haemoglobins, North Holland. Amsterdan, 1974.

2. Hegesh F, Hegesh J, Raftary A. Congenital methemoglobinemia with a deficiency of cytochrome b5. N Engl. J Med. 1986, 314:757-761.

3. Souza PC. Avaliação dos produtos de degradação oxidativa da hemoglobina S em eritrócitos de doentes falcêmicos. São José do Rio Preto, 1999. Dissertação de Mestrado em Ciências Biológicas - Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista.

4. Medeiros MHG, Bechara EJH, Naoum PC, Mourão CA. Oxygen toxicity and hemoglobinemia in subjects from a highly polluted town. Arch Environm Health. 1983, 38:11-16.

5. Moreira VA, Medeiros BC, Bonfim CMS, Pasquine R, Medeiros CR. Methemoglobinemia secundary to clofazimine treatment with chronic graft - host disease. Blood. 1998, 92:4872-4873.

6. Naoum PC. Hemoglobinopatias e Talassemias. Ed. Sarvier, São Paulo, 1997.

7. Bain B. Haemoglobinopathy diagnosis. Blackwell Science. Oxford, 2001.

8. Evelyn KA, Malloy HT. Microdetermination of oxyhemoglobin, methemoglobin and sulfhemoglobin in a single sample of blood. Journal of Biological Chemistry. 1938, 126:655-657.

9. Itano HA. Molar extinction coefficients of cyanmethemoglobin at 540 and 281nm. In: Izak G, Lewis SM, Modern Concepts in Hematology. New York. Academic Press, 28-8.