Hemoglobin and its derivatives: Difference between revisions
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== Hemoglobine in urine == | == 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. | 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: | |||
:::<math>\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}</math> | :::<math>\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}</math> | ||
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. | |||
[[Soubor:Oxidace tetrametylbenzidinu.png | center | 750px]] | [[Soubor:Oxidace tetrametylbenzidinu.png | center | 750px]] | ||
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 of occult bleeding in the digestive system'' (pdf) | ||
== [[Deriváty hemoglobinu]] == | == [[Deriváty hemoglobinu]] == | ||
Revision as of 17:49, 23 November 2021
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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.[1]
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:
- Anemia from increased blood loss:
- Acute blood loss.
- Chronic blood loss.
- 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).
- 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:
![{\displaystyle \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} }](https://wikimedia.org/api/rest_v1/media/math/render/svg/23377fd4d265df004d50c7657f43bcbbe4cdc8ff)
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 of occult bleeding in the digestive system (pdf)
Deriváty hemoglobinu
Úkol: Spektrofotometrické vyšetření hemoglobinu a jeho derivátů (pdf)
Úkol: Orientační stanovení karbonylhemoglobinu (pdf)
Glykovaný hemoglobin HBA1
Úkol: Stanovení glykovaného hemoglobinu (pdf)
Železo
__Železo
Úkol: Stanovení Fe v séru kolorimetrickou metodou (pdf)
