the microscope.
3. Haemochromogen
Haem and the ferrous porphyrin complexes react readily with basic substances such as hydrazines, primary amines, pyridines, or an imidazole such as the amino acid histidine, resulting compound is called a haemochromogen (haemochrome).
On spectroscopy, this compound shows:
(a) Soret band: A sharp absorption band near 400 mμ.
Note: It is a distinguishing feature of porphin ring and is characteristic of all porphyrins.
(b)In addition it shows two additional absorption bands: (i) ααααα-band: Narrow band at 559 mμ, and
(ii)βββββ-band: Broader band at 527 mμ. Both bands are in green part of the spectrum nearer to ‘D’ line.
4. Haematoporphyrin
It is a iron-free derivative. It can exist in two forms acid and alkaline. The derivative is prepared by mixing blood with sulphonic acid.
CLINICAL SIGNIFICANCE
Normal urine may contain trace amounts. It is found in the blood and urine in sulphonal poisoning and in certain cases of liver diseases.
5. Haematoidin
This compound is produced by the breakdown of Hb in the body. It is found as yellowish-red crystals in the region of old blood extravasation. Some authors believe that it is identical with bilirubin.
6. Methaemoglobin
It is a derivative in which Fe is in the ferric state and it is a true oxidation product of Hb. Increased amount of methaemoglobin in blood above normal is called as “methaemoglobinaemia”.
Causes: Methaemoglobin can be produced as follows: • In vitro by treating blood with potassium ferricyanide
• In vivo it is produced by certain oxidant drugs or exposure to certain poisons, e.g. chlorates, acetanilid, nitrites, nitrobenzene, antipyrin, phenacetin, sulphonal, and perhaps most important, the sulphonamide drugs.
CLINICAL IMPORTANCE
It is interesting to note that earlier treatments like use of AgNO3 solution in burn patients may produce the formation of methaemoglobin and toxic methaemoglobinaemia. Mechanism involves conversion of nitrate anion to “nitrite” by skin bacteria, which in turn converts Hb to methaemoglobin.
• Industrial hazard
– Nitrobenzene is used in manufacture of shoe dyes, floor polishes, cosmetics and explosives. Workers in these industries may be acutely or chronically poisoned if nitrobenzene is absorbed in sufficient amounts.
– Fumes from carbon arcs contain nitrous oxide which reacts with atmospheric O2 to form nitrogen dioxide. If this gas is breathed in high concen- tration methaemoglobin may be produced. • Familial methaemoglobinaemia
– An inherited disorder due to lack or absence of the enzyme methaemoglobin reductase, which is responsible for conversion of methaemoglobin to normal Hb (Fe++). In absence of the enzyme, methaemoglobin accumulates.
• Hb-M: Methaemoglobinaemia may also be found in individuals with abnormal haemoglobin as Hb-M.
Mechanism of Methaemoglobin Formation
1. In methaemoglobin formation, iron is oxidised to ferric state.
• As such it cannot bind O2
• The additional +ve charge per haem molecule is balanced with negative group, presumably –OH gr.
Differences Between Acid and Alkaline Haematin
Acid Haematin Alkaline Haematin
• Dilute HCl/Other acids split Hb into haem and globin, • Alkalies also split Hb into globin and “ferrohaem” haem is “ferrohaem”
• In presence of O2 haem is quickly oxidised to “ferri-haem” • In presence of O2 “ferrohaem” is converted to “ferri-haem” • Additional +ve charge is balanced by Cl– ion in case of • Additional +ve charge is balanced by –OH ion in case of
HCl and forms ‘acid haematin’ which is chemically, “Ferri- NaOH or KOH and forms alkaline haematin, which is
haem chloride” chemically “ferri-haem hydroxide”
Hb + HCl → globin + ferrohaem Hb + NaOH → globin + ferrohaem
2 ferro haem + 1/2 O2 + 2 HCl → 2 ferri-haem chloride 2 ferrohaem + ½ O2 + –OH ions → 2 ferri-haem hydroxide
(acid haematin) (alkali haematin)
• Absorption Bands • By spectroscopy: It shows a broader band at 600 mμ
By spectroscopy: It shows a thinner band at 650 mμ between C and D line, but near to D line. between C and D line, but nearer to C line.
SECTION TWO
Absorption spectra: By spectroscopy methaemoglobin gives characteristic absorption spectra.
1. Dilute neutral Met-Hb gives four absorption bands: (i) One broad band at 490 mμ in green part nearer to
‘F’ line
(ii) A narrow band at 540 mμ in green part
(iii) Another narrow band at 575 mμ in yellow part of the spectrum
(iv) A band at 634 mμ at red part of the spectrum. This band is the characteristic one and is used for detection of Met-Hb.
2. Alkaline Met-Hb gives only three bands: Narrow band at 490 mμ is missing.
7. Methaemalbumin
Methaemalbumin is formed by combination of free haematin (ferri-haem) with albumin. Normally methaemalbumin is not present in adult blood. It may be present at birth and can be detected in umbilical cord blood.
CLINICAL IMPORTANCE
Detection of methaemalbumin is an evidence of IV haemolysis as may occur in mismatched (incompatible) blood transfusion. Methaemalbumin can be detected by a very sensitive test called Schumm’s test.
Schumm’s Test
• A volume of plasma is covered with layer of ether • Add 1/10th volume or slightly more of conc. ammo-
nium sulphide (add from the side), mixed by shaking • The aqueous layer is taken out and examined spectro-
scopically.
Inference: If methaemalbumin is present an absorption band is seen at 558 mμ.
Toxic Effects of Met-Hb
Concentration Clinical features
in blood
10 to 20% Mild cyanosis
20 to 40% Visible cyanosis, fatigue and dyspnoea with activity
40 to 60% Produces severe cyanosis, severe cardiopulmonary symptoms, tachycardia, tachypnoea, and depression
> 60% Causes ataxia, severe cyanosis and dyspnoea, loss of consciousness and death.
2. During methaemoglobin formation, oxidation of Hb, mol O2 is changed to superoxide radical O’2 by univalent reduction. The superoxide is immediately destroyed by Cu-Zn containing enzyme superoxide dismutase. This enzyme helps to transfer electron from superoxide radical to H+ to produce H
2O2. H2O2 is then either broken down to O2 and H2O by catalase or, it is reduced to H2O by Se-containing enzyme glutathione peroxidase and reduced glutathione (G-S H).
Metabolism of Superoxide Radical
Reactions involved in metabolism of superoxide radical is shown below in the box.
Metabolism of Superoxide Radical
Mechanism of reconversion of methaemoglobin to Hb in normal health: In normal healthy adult, small amount of methaemoglobin may be present approx 0.3 gm/100 ml (about 1.7% of total Hb). This is converted to normal Hb by an enzyme called methaemoglobin reductase which requires NADH as coenzyme (Refer box below). An additional enzyme diaphorase I, has been found out, which is NADPH-dependant, can also perform the same function. Glutathione and ascorbic acid, reducing substances present in significant amounts in erythrocytes, may also be involved in reduction of Met-Hb.
Treatment
Principle: Injection of intravenous glucose or methylene blue, which helps to reduce Met-Hb (Fe+++) to Hb (Fe++), so that Hb is available again for O2 binding and transport. Methylene blue activates NADH or NADPH dependant methaemoglobin reductase/diaphorase I. Administration of ascorbic acid also helps in reduction.