Hemoglobin and its derivatives

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Hemoglobin is a red blood pigment that transports oxygen from the lungs to the tissues and transports CO2 and protons from peripheral tissues to the respiratory system.

náhled|Rozdíl mezi venózní a arteriální krví

The hemoglobin concentration in a healthy adult male is approximately 150 g / l, in an adult female about 140 g / l.  One gram of hemoglobin can bind up to 1.34 ml of oxygen.

Structure of hemoglobin

náhled|100px|Hemoglobin náhled|100px|Hem It is a tetrameric protein made up of four subunits. The two and two subunits are always identical.  There are four types of polypeptide chains, physiologically occurring hemoglobin, α, β, γ, and δ, which differ in the number and sequence of amino acids.  The tetramer consists of two α chains and two other types of chains that indicate the character of the whole hemoglobin molecule.  In adults, hemoglobin A predominates, with two β chains (146 amino acids) involved in addition to two α chains (141 amino acids).

Each subunit includes a polypeptide chain to which one heme is covalently attached.  The basis of the heme molecule is protoporphyrin, formed by 4 pyrrole nuclei connected by methenyl bridges with centrally bound iron.  Heme iron is a total of six bonds - it is connected to the nitrogen atoms of the pyrrole nuclei by four coordination bonds.  By another coordination valence, iron binds to the imidazole group of the amino acid histidine in the globin chain.  The sixth valence Fe is for the oxygen molecule (O₂).

Hemoglobin in the blood

Determination of hemoglobin in the blood is one of the most basic laboratory tests.  Blood hemoglobin is the main criterion for assessing whether it is anemia.  The term anemia is used when hemoglobin or erythrocytes fall below the lower limit of physiological levels. Anemia is a very common clinical finding.  This is a condition that leads to a reduction in oxygen binding capacity and a consequent tissue respiratory disorder.

Causes of Anemia

Anemia occurs when erythropoiesis is unable to meet the requirements for new red blood cells.  It develops as a result of blood loss or increased loss of red blood cells or insufficient red blood cell production.  The following is a list of some specific causes of anemia:

1. Anemia from increased blood loss:
   • Acute blood loss.
   • Chronic blood loss.
2. Anemia due to increased erythrocyte breakdown (hemolytic conditions).
   • Autoimmune hemolytic anemia (presence of antibodies against own erythrocytes).
   • Erythrocyte membrane disorder (deviation in erythrocyte membrane composition).
   • Hereditary erythrocyte enzyme defects (pyruvate kinase, glucose-6-phosphate dehydrogenase).
   • Unstable hemoglobin - hemoglobinopathies (e.g. hemoglobin S in sickle cell disease).
3. Anemia from decreased erythrocyte production:
   • Lack of substances needed for erythropoiesis (iron deficiency, vitamin B12 deficiency, folic acid deficiency, erythropoietin deficiency - chronic renal diseases, lack of other substances such as vitamins B1, B6).
   • Anemia due to chemical, physical and radiation damage.
   • Anemia in chronic inflammatory, infectious and cancerous diseases.

Elevated hemoglobin levels may be a sign of dehydration or chronic decreased pulmonary ventilation.  Rarely, it can be caused by some myeloproliferative conditions, such as polycythemia vera.

Principle of hemoglobin determination in blood

Oxidation of hemoglobin to methemoglobin:

HbFeII	+	[FeIII(CN)6]3−	→	HbFeIII	+	[FeII(CN)6]4−
Hemoglobin				Methemoglobin

Conversion of methemoglobin to cyanomethemoglobin:

HbFeIII	+	CN−	→	HbFeIIICN
Methemoglobin				Cyanomethemoglobin

The photometric determination is based on the oxidation of ferrous iron in hemoglobin with potassium ferrocyanide to ferric iron.  The resulting methemoglobin is converted to a very stable cyanomethemoglobin in a further reaction with potassium cyanide with a single broad absorption maximum in the visible region at 540 nm.

Assessment: The reference range for hemoglobin in the blood (B hemoglobin) for an adult male is 130-180 g / l and for a female 120-160 g / l.

Task: Determination of hemoglobin in the blood (pdf)

Hemoglobine in urine

Up to a million erythrocytes per day are excreted in the urine of completely healthy people.  This very small amount cannot be demonstrated by conventional chemical tests.  Occurrence of a larger number of erythrocytes (hematuria, erythrocyturia) or penetration of free hemoglobin, or  muscle myoglobin, into definitive urine (hemoglobinuria or myoglobinuria) is almost always a pathological finding.  We observe macroscopic hematuria with the naked eye;  the urine is pinkish (comparable to water from washed meat) and hemoglobin can be detected spectroscopically in it.  There is at least 1 g of hemoglobin per liter in the urine.  In massive hemoglobinuria, the urine may have a colour of a dark beer (degradation of hemoglobin to hematin).  Microscopic hematuria can only be detected biochemically.

Determination of hemoglobin in urine

Hemoglobin catalyzes, like peroxidase, the oxidation (dehydrogenation) of some substrates (eg benzidine derivatives) by hydrogen peroxide:

$\mathrm {H} _{2}\mathrm {O} _{2}+\mathrm {H} _{2}\mathrm {A} \ {\xrightarrow[{\mathrm {nebo\ hemoglobin\ a\ jin{\acute {e}}\ l{\acute {a}}tky} }]{\mathrm {peroxid{\acute {a}}zy} }}\ 2\ \mathrm {H} _{2}\mathrm {O} +\mathrm {A}$

However, it is not an enzyme activity (catalysis is conditioned by heme iron) and therefore it is not lost even after heat denaturation.  We are talking about pseudoperoxidase activity, which is used for sensitive but non-specific evidence of hemoglobin or trace amounts of blood.  It is preferable to use a chromogenic substrate to monitor the reaction, i.e., a substance that provides a markedly colored product by dehydrogenation (often benzidine or its non-carcinogenic derivatives, aminophenazone, etc.).

The reagent zone of the diagnostic stripes contains a chromogen (eg tetramethylbenzidine) with stabilized hydrogen peroxide (eg cumene hydroperoxide).  In the presence of free hemoglobin (hemoglobinuria), the indication zone turns uniformly blue.  If erythrocytes (erythrocyturia) are present in the urine, intensely green-blue dots to spots form.

Hemoglobinuria can be encountered in intravascular hemolysis.  Damage to the glomerular membrane (glomerular hematuria) and bleeding from any part of the urinary tract lead to more frequent erythrocyturia.  It is often found in urinary tract infections, urolithiasis and urogenital tract tumors.

In addition to hemoglobin, myoglobin also provides a pseudoperoxidase response, which can be excreted in the urine during skeletal muscle breakdown (rhabdomyolysis, crush syndrome).  The positivity of the test may also be due to peroxidases of leukocytes or certain bacteria, yeasts or fungi, which may occur in the urine, especially in urinary tract infections.  To rule out the possibility of a false positive reaction due to cellular peroxidases, the reaction must be performed with boiled urine.

Contamination of the sampling vessel with strong oxidizing agents also causes a false positive reaction.  On the other hand, the presence of strong reducing substances (eg ascorbic acid) can slow down or even stop the pseudoperoxidase reaction and thus cause false negative results.

Assesment: Determination of blood and hemoglobin in urine (pdf)

Hemoglobin in stool - occult bleeding

Demonstration of occult (hidden) bleeding is used to detect the early stages of colorectal cancer, when radical and effective treatment is possible.  The examination consists of capturing traces of blood in the stool, using various methodological procedures:

• The methods use the pseudoperoxidase activities of hemoglobin.  The patient must maintain a diet for 3 days before the examination, exclude uncooked meat, salami, bananas, tomatoes from the diet, and must not take drugs containing ascorbic acid or acetylsalicylic acid. The patient then takes samples from three consecutive stools and applies them to the test cards.  The evaluation is performed in the laboratory, the principle is similar to the hemoPHAN diagnostic stripes.
• Other methods are based on the immunochemical detection of hemoglobin with an anti-human hemoglobin antibody. Immunochemical methods are more sensitive and specific, there is no need to follow a diet before the examination.  Positive results must be verified by other diagnostic methods.

Assesment: Test for occult bleeding in the digestive tract (pdf)

Hemoglobin derivatives

Hemoglobin derivatives include the following types:

Oxyhemoglobin and deoxyhemoglobin

Oxygen-carrying hemoglobin is referred to as oxyhemoglobin (oxyHb).  Each Hb molecule can bind 4 molecules of oxygen.  After the release of oxygen, we speak of deoxyhemoglobin (deoxyHb).  In both forms, iron is divalent because only FeII + -containing hemoglobin can reversibly bind and transport the oxygen molecule.  Oxygenation of the hemoglobin molecule changes the electronic state of the FeII + -hem complex, which results in a change in the color of the dark red (typical of venous blood) to a bright red color (arterial blood).  In the human body, about 98.5% [2] of oxygen is bound to hemoglobin.

Carbaminohemoglobin

Carbaminohemoglobin is a hemoglobin to which CO2 is bound.  Carbon dioxide binds to the globin chain of hemoglobin.  The binding of CO2 to hemoglobin reduces the affinity of hemoglobin for oxygen.

Methemoglobin

Methemoglobin (metHb; also hemiglobin or ferihemoglobin [1]) is characterized by the presence of ferric iron, which is formed by the oxidation of ferrous iron in hemoglobin [3].  Methemoglobin loses its ability to reversibly bind oxygen.  In its place, Fe^III+ binds a water molecule through the sixth coordination bond.  The color of methemoglobin is chocolate brown. Methemoglobin is also present physiologically in small amounts in erythrocytes (about 1–3% of the total hemoglobin concentration [4]).  This is mainly due to the effect of nitrite, which is formed from nitrates contained in food.  The reverse reduction of methemoglobin to hemoglobin is mainly ensured by NADH-dependent cytochrome-b₅ reductase (also methemoglobin reductase).  A minor role is played by NADPH-dependent methemoglobin reductase, which is dependent on the supply of NADPH from the pentose cycle and on the presence of another electron transporter (eg flavin). [5]  Non-enzymatic mechanisms include the action of glutathione and ascorbic acid.

Elevated blood levels of methemoglobin are called methemoglobinaemia.  The causes are different:

• Hereditary methemoglobinemia is usually caused by a congenital defect of NADH-dependent methemoglobin reductase or the presence of abnormal hemoglobin M.
• Acquired methemoglobinemia is the most common form of methemoglobinemia.  May be caused by oxidizing agents [6]:
  • poisoning by certain substances (nitrobenzene, aniline and its derivatives - eg some dyes),
  • by the action of some drugs (local anesthetics - benzocaine, then phenacetin, sulfonamides),
  • increased content of nitrates and nitrites in water and food.

Newborns are particularly sensitive to the increased content of these substances due to the immaturity of the reduction systems and the increased proportion of fetal hemoglobin, which is more easily oxidized.  Methemoglobinemia is manifested by cyanosis with a characteristic gray-brown tint and hypoxia.

Symptoms of methemoglobinemia

Methemoglobin values	Symptoms
0–2 %	normal value
< 10 %	cyanosis
< 35 %	cyanosis and other symptoms (headache, dyspnoea)
70 %	lethal concentration

Part of the therapy of acquired methemoglobinemia is the administration of some reducing agents - methylene blue or ascorbic acid.

Carbonyl hemoglobin

Carbonylhemoglobin (COHb, carboxyhemoglobin) is formed by the binding of carbon monoxide to hemoglobin.  The bond formed is 250-300 times stronger than the oxygen bond.  Carbonyl hemoglobin cannot transport oxygen and cellular hypoxia develops due to the blood's reduced ability to carry oxygen.  In excess oxygen, the binding of carbon monoxide to hemoglobin is reversible.  Therefore, inhalation of O₂ is most important in carbon monoxide poisoning.

COHb can also occur in small amounts in healthy people.  For urban dwellers, values ​​of around 2% are evident; for heavy smokers, COHb can rise to as much as 10% of total hemoglobin.  Staying in an environment containing 0.1% CO for several minutes can increase the carbonyl hemoglobin concentration to 50%.

Carbon monoxide is formed during imperfect combustion of fuels, it is also contained in exhaust gases and in smoke during fires in closed rooms.

Symptoms of carbon monoxide poisoning

COHb values in %	Symptoms
10	more exertion shortness of breath
20–40	headache, shortness of breath, fatigue, vomiting
40–60	hyperventilation, tachycardia, syncope, convulsions
60–80	coma, death

Carbonyl hemoglobin is crimson red;  even people with severe carbon monoxide poisoning tend to have "healthy" pink skin.  Compared to hemoglobin, carbonyl hemoglobin is more resistant to chemical influences, it changes more slowly due to the action of various agents.

Úkol: Spektrofotometrické vyšetření hemoglobinu a jeho derivátů (pdf)

Úkol: Orientační stanovení karbonylhemoglobinu (pdf)

Glycated hemoglobin HBA₁

Glycated hemoglobin is formed by a non-enzymatic reaction between hemoglobin and blood glucose.  Its creation is irreversible.

__Glykovaný hemoglobin

Úkol: Stanovení glykovaného hemoglobinu (pdf)

Železo

__Železo

Úkol: Stanovení Fe v séru kolorimetrickou metodou (pdf)

Odkazy

Související články

• Karbonylhemoglobin
• Karbaminohemoglobin
• Methemoglobin
• Vyšetrenie hemoglobínu a jeho metabolizmu
• Hem

Reference

Kategorie:Biochemie Kategorie:Fyziologie