Carbonic anhydrase(synonym: carbonate dehydratase, carbonate hydrolyase) is an enzyme that catalyzes the reversible reaction of carbon dioxide hydration: CO 2 + H 2 O Û H 2 CO 3 Û H + + HCO 3. Contained in red blood cells, cells of the gastric mucosa, adrenal cortex, kidneys, and in small quantities in the central nervous system, pancreas and other organs. The role of carbonic anhydrase in the body is associated with maintaining acid-base balance, transport of CO 2, formation of hydrochloric acid by the gastric mucosa. The activity of carbonic anhydrase in the blood is normally quite constant, but in some pathological conditions it changes dramatically. An increase in carbonic anhydrase activity in the blood is observed in anemia of various origins, circulatory disorders of the II-III degree, some lung diseases (bronchiectasis, pneumosclerosis), as well as during pregnancy. A decrease in the activity of this enzyme in the blood occurs with acidosis of renal origin, hyperthyroidism. With intravascular hemolysis, carbonic anhydrase activity appears in the urine, while normally it is absent. It is advisable to monitor the activity of carbonic anhydrase in the blood during surgical interventions on the heart and lungs, because it can serve as an indicator of the body's adaptive capabilities, as well as during therapy with carbonic anhydrase inhibitors - hypothiazide, diacarb.
To determine the activity of carbonic anhydrase, radiological, immunoelectrophoretic, colorimetric and titrimetric methods are used. The determination is made in whole blood taken with heparin or in hemolyzed red blood cells. For clinical purposes, the most acceptable colorimetric methods for determining carbonic anhydrase activity (for example, modifications of the Brinkman method), based on determining the time required to shift the pH of the incubation mixture from 9.0 to 6.3 as a result of CO 2 hydration. Water saturated with carbon dioxide is mixed with an indicator-buffer solution and a certain amount of blood serum (0.02 ml) or a suspension of hemolyzed erythrocytes. Phenol red is used as an indicator. As carbonic acid molecules dissociate, all new CO 2 molecules undergo enzymatic hydration. To obtain comparable results, the reaction must always proceed at the same temperature; it is most convenient to maintain the temperature of melting ice at 0°. The control reaction time (spontaneous reaction of CO 2 hydration) is normally 110-125 With. Normally, when determined by this method, the activity of carbonic anhydrase is on average 2-2.5 conventional units, and in terms of 1 million red blood cells it is 0.458 ± 0.006 conventional units (a unit of carbonic anhydrase activity is taken to be a 2-fold increase in the rate of the catalyzed reaction).
Bibliography: Clinical evaluation of laboratory tests, ed. WELL. Titsa, per. from English, p. 196, M., 1986.
1The purpose of the work is to determine the factors influencing the activity of zinc-containing carbonic anhydrase in the reproductive system of male rats under conditions of exposure to low-intensity microwave radiation. Carbonic anhydrase plays an important role in the metabolism of seminal plasma and sperm maturation. Carbonic anhydrase activity in water-salt extracts of epididymis and testes of rats in the control group, according to our data, ranges from 84.0 ± 74.5 U/ml, which in terms of tissue weight is 336.0 ± 298.0 U/mg. The relationship between the concentration of zinc and polyamine ions and the activity of carbonic anhydrase was studied. The activity of carbonic anhydrase in the reproductive system of male rats has a complex regulation scheme, which obviously is not limited to the factors we have described. Based on the results obtained, it can be concluded that the role of various regulators of the activity of this enzyme varies depending on the degree of carbonic anhydrase activity. It is likely that high spermine concentrations limit the transcription of the carbonic anhydrase gene, given the data on the functions of this polyamine. Spermidine probably serves as a limiting factor at the post-tribosomal stages of regulation of carbonic anhydrase activity, and putrescine and the concentration of zinc ions are interrelated activation factors.
reproductive system of male rats
zinc ion concentration
polyamines
carbonic anhydrase
1. Boyko O.V. Methodological aspects of the use of hydrochloric acid spermine and spermidine for the identification of uropathogenic microflora / O.V. Boyko, A.A. Terentyev, A.A. Nikolaev // Problems of reproduction. – 2010. – No. 3. – P. 77-79.
2. Ilyina O.S. Changes in the zinc content in human blood in type I diabetes mellitus and features of the hypoglycemic effect of the zinc-containing insulin-chondroitin sulfate complex: abstract. dis. ...cand. biol. Sci. – Ufa, 2012. – 24 p.
3. Lutsky D.L. Protein spectrum of ejaculates of different fertility / D.L. Lutsky, A.A. Nikolaev, L.V. Lozhkina // Urology. – 1998. – No. 2. – P. 48-52.
4. Nikolaev A.A. Activity of spermoplasmic enzymes in ejaculates of different fertility / A.A. Nikolaev, D.L. Lutsky, V.A. Bochanovsky, L.V. Lozhkina // Urology. – 1997. – No. 5. – P. 35.
5. Ploskonos M.V. Determination of polyamines in various biological objects / M.V. Ploskonos, A.A. Nikolaev, A.A. Nikolaev // Astrakhan State. honey. acad. – Astrakhan, 2007. – 118 p.
6. Polunin A.I. The use of zinc preparation in the treatment of male subfertility / A.I. Polunin, V.M. Miroshnikov, A.A. Nikolaev, V.V. Dumchenko, D.L. Lutsky // Microelements in medicine. – 2001. – T. 2. – No. 4. – P. 44-46.
7. Haggis G.C., Gortos K. Carbonic anhydrase activity of the reproductive tract tissues of male rats and its relationship to semen production // J. Fert. Reprod. – 2014. - V. 103. - P. 125-130.
It is known that the activity of zinc-containing carbonic anhydrase is high in the reproductive system of male birds, mammals and humans. The activity of this enzyme influences the maturation of sperm, their number and sperm volume. But there is no information about changes in carbonic anhydrase activity under the influence of other constant components of the reproductive system, such as zinc ions and polyamines (putrescine, spermine and spermidine), which actively influence spermatogenesis. Only a general description of the consequences of changes in carbonic anhydrase activity on the morphofunctional state of the organs of the reproductive system of male rats, the number of sperm, and their motility is given.
The purpose of our work was a study of the activity of zinc-containing carbonic anhydrase and its relationship with the level of polyamines and zinc ions in the tissue of the reproductive system of sexually mature male rats.
Materials and methods. The experimental part of the study included 418 male white Wistar rats. The rats were 6-7 months old (mature individuals). The body weight of the rats was 180-240 g, kept under standard vivarium conditions. To avoid the influence of seasonal differences in responses to experimental influences, all studies were carried out in the autumn-winter period of the year. The collection of testes and epididymis from rats was carried out under ether anesthesia (experimental studies were carried out in strict accordance with the Declaration of Helsinki on the Humane Treatment of Animals).
The objects of our study were water-salt extracts of epididymis and testes of sexually mature male white rats. Extracts were prepared in Tris-hydrochloric acid buffer pH = 7.6 in a weight/volume ratio of 1/5, after four times freezing, thawing and centrifugation at 8000 g for 50 minutes, the samples were frozen and stored at -24 °C until the study.
Determination of zinc. To 2 ml of the extract under study, 0.1 ml of 10% NaOH and 0.2 ml of a 1% solution of dithizone in carbon tetrachloride were added. In the negative control, 2 ml of distilled water was added, in the positive control - 2 ml of a 20 μmol zinc sulfate solution (molar concentration of a standard zinc sulfate solution). Samples were photometered at 535 nm. The concentration of zinc cations in the sample was calculated using the formula: CZn=20 µmol × Sample OD535/Standard OD535, where Sample OD535 is the optical density of the sample, measured at 535 nm; OD535 Standard - optical density of a standard 20 micromolar solution of zinc sulfate, measured at 535 nm.
Determination of carbonic anhydrase. The method is based on the reaction of bicarbonate dehydration with the removal of carbon dioxide formed as a result of dehydration with intensive bubbling of the reaction medium with air freed from carbon monoxide and simultaneous recording of the rate of change in pH. The reaction is initiated by quickly introducing a solution of the substrate - sodium bicarbonate (10 mM) into the reaction mixture containing the test sample. In this case, the pH increases by 0.01-0.05 units. Samples (10.0-50.0 mg) of epididymis and testes of sexually mature male white rats were homogenized and centrifuged at 4500 g for 30 minutes. at 4 °C, and the supernatant is diluted with double distilled water at 4 °C to a volume that would allow the reaction time to be measured. Carbonic anhydrase activity is determined by the change in the initial pH value from 8.2 to 8.7 in the CO2 dehydration reaction. The rate of accumulation of hydroxyl ions is measured electrometrically using a sensitive programmable pH meter (InoLab pH 7310) interfaced with a PC. The pH shift from 8.2 to 8.7, as a function of time in the linear section, takes into account the enzyme activity. The average time (T) for 4 measurements was calculated. The time of pH change during spontaneous hydration of CO2 in a medium without a sample was taken as control. Carbonic anhydrase activity was expressed in enzyme units (U) per mg of wet tissue according to the equation: ED = 2 (T0 - T)/ (T0 × mg tissue in the reaction mixture), where T0 = average time for 4 measurements of a pure solution of 4 ml of cooled, saturated carbon dioxide, bidistilled water.
Determination of polyamines. Samples (100–200 mg) of epididymis and testes of mature male albino rats were homogenized, suspended in 1 ml of 0.2 normal perchloric acid to extract free polyamines, and centrifuged. To 100 μl of the supernatant, 110 μl of 1.5 M sodium carbonate and 200 μl of dansyl chloride (7.5 mg/ml solution in acetone; Sigma, Munich, Germany) were added. In addition, 10 μL of 0.5 mM diaminohexane was added as an internal standard. After 1 h of incubation at 60°C in the dark, 50 μL of proline solution (100 mg/mL) was added to bind free dansyl chloride. Then dansyl derivatives of polyamines (hereinafter referred to as DNSC-polyamines) were extracted with toluene, sublimated in a vacuum evaporator and dissolved in methanol. Chromatography was performed on a reverse phase LC 18 column (Supelco), in a high performance liquid chromatography system (Dionex) consisting of a gradient mixer (model P 580), an automatic injector (ASI 100) and a fluorescence detector (RF 2000). Polyamines were eluted in a linear gradient from 70% to 100% (v/v) methanol in water at a flow rate of 1 mL/min and detected at an excitation wavelength of 365 nm and an emission wavelength of 510 nm. Data were analyzed using Dionex Chromeleon software and quantification was performed with calibration curves obtained from a mixture of pure substances (Figure A).
High performance chromatography of DNSC polyamines:
A - chromatogram of a standard mixture of DNSC-polyamines; B - chromatogram of DNSC-polyamines from one of the tissue samples of the epididymis and testes of male rats. 1 - putrescine; 2 - cadaverine; 3 - hexanediamine (internal standard); 4 - spermidine; 5 - spermine. The x-axis is time in minutes, the y-axis is fluorescence. Unnumbered peaks - unidentified impurities
Research results and discussion. As is known, carbonic anhydrase plays an important role in the metabolism of seminal plasma and sperm maturation. Carbonic anhydrase activity in water-salt extracts of epididymis and testes of rats in the control group, according to our data, ranges from 84.0 ± 74.5 U/ml, which in terms of tissue weight is 336.0 ± 298.0 U/mg. Such a high activity of the enzyme can be explained by its important physiological role. For comparison, the level of activity of this enzyme in other tissues of the same animals is much lower (Table 1), except for whole blood, in which high activity of erythrocyte carbonic anhydrase is known. However, what is noteworthy is the very wide scatter in the values of carbonic anhydrase activity in epididymis and testes, the coefficient of variation of which is more than 150% (Table 1).
Table 1
Carbonic anhydrase activity in tissues of sexually mature males
|
Male rat tissue |
Enzyme activity, units |
Number of observations |
The coefficient of variation, % |
|
brain tissue |
|||
|
Muscle |
|||
|
Mucosa of the gastrointestinal tract |
|||
|
epididymis and testes |
|||
|
Whole blood |
This indicates the influence of unaccounted factors on the enzyme activity. There are two circumstances that explain this feature. Firstly, it is known that biologically active amines, including the polyamines spermidine and spermine, are capable of activating carbonic anhydrase. It is the male reproductive system that is the richest source of spermine and spermidine. Therefore, we carried out a parallel determination of the concentration of polyamines in water-salt extracts of epididymis and testes of male rats. The polyamines spermidine, spermine, and putrescine were analyzed by HPLC as described in Methods. It was shown that spermine, spermidine and putrescine were detected in the tissue of the epididymis and testes of male rats (Fig. B).
In healthy sexually mature male rats, the level of spermine was 5.962±4.0.91 µg/g tissue, spermidine 3.037±3.32 µg/g tissue, putrescine 2.678±1.82 µg/g tissue, and spermine/spermidine ratio 1.88- 2.91. Moreover, according to our data, both the level of spermidine and the level of spermine (to a lesser extent) are subject to significant fluctuations. Correlation analysis showed a significant positive relationship (r=+0.3) between the levels of spermine and spermidine, and, respectively, spermidine and putrescine (r=+0.42). Apparently, this circumstance is one of the factors influencing the high dispersion of the results of determining carbonic anhydrase activity.
Another regulator of carbonic anhydrase activity may be the level of zinc in the reproductive tissue of sexually mature male rats. According to our data, the level of zinc ion varies widely, from 3.2 to 36.7 μg/g of tissue of the total preparation of the testes and epididymis of sexually mature male rats.
Correlation analysis of zinc levels with levels of spermine, spermidine and carbonic anhydrase activity showed different levels of positive correlation between the concentration of zinc ions and these metabolites. An insignificant level of association was found with spermine (+0.14). Given the number of observations used, this correlation is not significant (p≥0.1). A significant positive correlation was found between the level of zinc ions and the concentration of putrescine (+0.42) and the concentration of spermidine (+0.39). An expectedly high positive correlation (+0.63) was also found between the concentration of zinc ions and carbonic anhydrase activity.
At the next stage, we tried to combine the concentration of zinc and the level of polyamines as factors regulating carbonic anhydrase activity. When analyzing the variation series of the joint determination of the concentration of zinc ions, polyamines and carbonic anhydrase activity, some regularities were revealed. It was shown that out of 69 studies conducted on the level of carbonic anhydrase activity, three groups can be distinguished:
Group 1 - high activity from 435 to 372 units (number of observations 37),
Group 2 - low activity from 291 to 216 units (number of observations 17),
Group 3 - very low activity from 177 to 143 units (number of observations 15).
When ranking the levels of polyamines and the concentration of zinc ions with these groups, an interesting feature was revealed that did not appear when analyzing the variation series. The maximum spermine concentrations (on average 9.881±0.647 μg/g tissue) are associated with the third group of observations with very low carbonic anhydrase activity, and the minimum (on average 2.615±1.130 μg/g tissue) with the second group with low enzyme activity.
The largest number of observations is associated with the first group with a high level of carbonic anhydrase activity; in this group, spermine concentrations are close to average values (on average 4.675 ± 0.725 μg/g of tissue).
The concentration of zinc ions exhibits a complex relationship with the activity of carbonic anhydrase. In the first group of carbonic anhydrase activity (Table 2), the concentration of zinc ions is also higher than the values in other groups (on average 14.11±7.25 μg/g of tissue). Further, the concentration of zinc ions decreases in accordance with the decrease in carbonic anhydrase activity, but this decrease is not proportional. If in the second group the activity of carbonic anhydrase decreases compared to the first by 49.6% and in the third by 60.35%, then the concentration of zinc ions decreases in the second group by 23%, and in the third by 39%.
table 2
The relationship between the concentration of polyamines and zinc ions and the activity of carbonic anhydrase
|
Activity groups carbonic anhydrase, units |
Average concentration spermine, µg/g tissue |
Average concentration spermidine, µg/g tissue |
Average concentration putrescine, µg/g tissue |
Average concentration zinc ions, µg/g tissue |
This indicates additional factors influencing the activity of this enzyme. The dynamics of putrescine concentration looks somewhat different (Table 2). The level of this polyamine is falling at a faster pace, and in the third comparison group the level of putrescine is lower on average by almost 74%. The dynamics of the spermidine level differs in that the “jumping” concentration values of this polyamine are associated primarily with the second group of carbonic anhydrase activity levels. With high activity of this enzyme (group 1), the spermidine concentration is slightly higher than the average for all observations, and in the third group it is almost 4 times lower than the concentration in the second group.
Thus, the activity of carbonic anhydrase in the reproductive system of male rats has a complex regulation scheme, which obviously is not limited to the factors we have described. Based on the results obtained, it can be concluded that the role of various regulators of the activity of this enzyme varies depending on the degree of carbonic anhydrase activity. It is likely that high spermine concentrations limit the transcription of the carbonic anhydrase gene, given the data on the functions of this polyamine. Spermidine probably serves as a limiting factor at the post-tribosomal stages of regulation of carbonic anhydrase activity, and putrescine and the concentration of zinc ions are interrelated activation factors.
Under these conditions, assessing the influence of external factors (including those changing reproductive function) on the activity of carbonic anhydrase, as one of the important links in the metabolism of the reproductive system of male mammals, becomes not only important, but also a rather complex process, requiring a large number of controls and multilateral assessment.
Bibliographic link
Kuznetsova M.G., Ushakova M.V., Gudinskaya N.I., Nikolaev A.A. REGULATION OF THE ACTIVITY OF ZINC-CONTAINING CARBONAN HYDRASE IN THE REPRODUCTIVE SYSTEM OF MALE RATS // Modern problems of science and education. – 2017. – No. 2.;URL: http://site/ru/article/view?id=26215 (date of access: 07/19/2019).
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55-58 vol.% of carbon dioxide can be extracted from venous blood. Most of the CO2 extracted from the blood comes from carbonic acid salts present in plasma and erythrocytes, and only about 2.5 vol.% of carbon dioxide is dissolved and about 4-5 vol.% is combined with hemoglobin in the form of carbohemoglobin.
Carbonic acid is formed from carbon dioxide in red blood cells, which contain the enzyme carbonic anhydrase, which is a powerful catalyst that accelerates the hydration reaction of CO2.
Binding of carbon dioxide in the blood in the capillaries of the systemic circle. Carbon dioxide formed in the tissues diffuses into the blood of the blood capillaries, since the CO2 tension in the tissues significantly exceeds its tension in the arterial blood. CO2 dissolved in plasma diffuses into the red blood cell, where under the influence carbonic anhydrase it instantly turns into carbonic acid,
According to calculations, the activity of carbonic anhydrase in erythrocytes is such that the reaction of carbon dioxide hydration is accelerated by 1500-2000 times. Since all the carbon dioxide inside the erythrocyte is converted into carbonic acid, the CO2 tension inside the erythrocyte is close to zero, so more and more new amounts of CO2 enter the erythrocyte. Due to the formation of carbonic acid from CO3 in the erythrocyte, the concentration of HCO3" ions increases, and they begin to diffuse into the plasma. This is possible because the surface membrane of the erythrocyte is permeable to anions. For cations, the erythrocyte membrane is practically impermeable. Instead of HCO3" ions, the erythrocyte ion enters chlorine The transition of chlorine ions from the plasma into the erythrocyte releases sodium ions in the plasma, which bind the HCO3 ions entering the erythrocyte, forming NaHCO3. Chemical analysis of venous blood plasma shows a significant increase in bicarbonate in it.
The accumulation of anions inside the erythrocyte leads to an increase in osmotic pressure inside the erythrocyte, and this causes the passage of water from the plasma through the surface membrane of the erythrocyte. As a result, the volume of red blood cells in the systemic capillaries increases. A study using hematocrit revealed that red blood cells occupy 40% of the volume of arterial blood and 40.4% of the volume of venous blood. It follows from this that the volume of venous blood erythrocytes is greater than that of arterial erythrocytes, which is explained by the penetration of water into them.
Simultaneously with the entry of CO2 into the erythrocyte and the formation of carbonic acid in it, oxygen is released from oxyhemoglobin and converted into reduced hemoglobin. The latter is a much less dissociating acid than oxyhemoglobin and carbonic acid. Therefore, when oxyhemoglobin is converted into hemoglobin, H2CO3 displaces potassium ions from hemoglobin and, combining with them, forms the potassium salt of bicarbonate.
The liberated H˙ ion of carbonic acid binds to hemoglobin. Since reduced hemoglobin is a slightly dissociated acid, there is no acidification of the blood and the difference in pH between venous and arterial blood is extremely small. The reaction occurring in the red blood cells of tissue capillaries can be represented as follows:
KHbO2 + H2CO3= HHb + O2 + KHSO3
From the above it follows that oxyhemoglobin, turning into hemoglobin and giving up the bases associated with it to carbon dioxide, promotes the formation of bicarbonate and the transport of carbon dioxide in this form. In addition, gcmoglobin forms a chemical compound with CO2 - carbohemoglobin. The presence of hemoglobin and carbon dioxide in the blood was determined by the following experiment. If potassium cyanide is added to whole blood, which completely inactivates carbonic anhydrase, it turns out that the red blood cells of such blood bind more CO2 than plasma. From this it was concluded that the binding of CO2 by erythrocytes after inactivation of carbonic anhydrase is explained by the presence of a hemoglobin compound with CO2 in erythrocytes. It was later discovered that CO2 attaches to the amine group of hemoglobin, forming a so-called carbamine bond.
The reaction of carbohemoglobin formation can go in one direction or the other depending on the tension of carbon dioxide in the blood. Although a small part of the total amount of carbon dioxide that can be extracted from the blood is combined with hemoglobin (8-10%), the role of this compound in the transport of carbon dioxide in the blood is quite large. Approximately 25-30% of the carbon dioxide absorbed by the blood in the systemic capillaries combines with hemoglobin to form carbohemoglobin.
Release of CO2 by blood in the pulmonary capillaries. Due to the lower partial pressure of CO2 in the alveolar air compared to its tension in the venous blood, carbon dioxide passes through diffusion from the blood of the pulmonary capillaries into the alveolar air. The CO2 tension in the blood drops.
At the same time, due to the higher partial pressure of oxygen in the alveolar air compared to its tension in the venous blood, oxygen flows from the alveolar air into the blood of the capillaries of the lungs. The O2 tension in the blood increases, and hemoglobin is converted into oxyhemoglobin. Since the latter is an acid, the dissociation of which is much higher than that of carbonic acid hemoglobin, it displaces carbonic acid from its potassium acid. The reaction goes as follows:
ННb + O2 + KНSO3= KНbO2+H2CO3
Carbonic acid, freed from its bond with bases, is broken down by carbonic anhydrase into carbon dioxide into water. The importance of carbonic anhydrase in the release of carbon dioxide in the lungs can be seen from the following data. In order for the dehydration reaction of H2CO3 dissolved in water to occur, with the formation of the amount of carbon dioxide that leaves the blood while it is in the capillaries of the lungs, it takes 300 seconds. Blood passes through the capillaries of the lungs within 1-2 seconds, but during this time, dehydration of carbonic acid inside the red blood cell and diffusion of the resulting CO2 first into the blood plasma and then into the alveolar air.
Since the concentration of HCO3 ions in erythrocytes decreases in the pulmonary capillaries, these ions from the plasma begin to diffuse into the erythrocytes, and chloride ions diffuse from the erythrocytes into the plasma. Due to the fact that the tension of carbon dioxide in the blood of the pulmonary capillaries decreases, the carbamine bond is cleaved and carbohemoglobin releases carbon dioxide.
Dissociation curves of carbonic acid compounds in the blood. As we have already said, over 85% of the carbon dioxide that can be extracted from the blood by acidifying it is released as a result of the breakdown of bicarbonates (potassium in red blood cells and sodium in plasma).
The binding of carbon dioxide and its release into the blood depend on its partial tension. It is possible to construct dissociation curves for carbon dioxide compounds in the blood, similar to the dissociation curves for oxyhemoglobin. To do this, the volume percentages of carbon dioxide bound in blood are plotted along the ordinate axis, and the partial stresses of carbon dioxide are plotted along the abscissa axis. The lower curve in Fig. 58 shows the binding of carbon dioxide by arterial blood, the hemoglobin of which is almost completely saturated with oxygen. The upper curve shows the binding of acid gas by venous blood.
The difference in the height of these curves depends on the fact that arterial blood, rich in oxyhemoglobin, has a lower ability to bind carbon dioxide compared to venous blood. Being a stronger acid than carbonic acid, oxyhemoglobin removes bases from bicarbonates and thereby contributes to the release of carbonic acid. In tissues, oxyhemoglobin, turning into hemoglobin, gives up the bases associated with it, increasing the binding of acid gas in the blood.
Point A on the lower curve in Fig. 58 corresponds to an acid voltage of 40 mm Hg. Art., i.e. the voltage that actually exists in the arterial blood. At this voltage, 52 vol.% CO2 is bound. Point V on the upper curve corresponds to an acid gas voltage of 46 mmHg. Art., i.e. actually present in the venous blood. As can be seen from the curve, at this voltage, venous blood binds 58 vol.% carbon dioxide. The AV line connecting the upper and lower curves corresponds to those changes in the ability to bind carbon dioxide that occur when arterial blood is converted into venous or, conversely, venous blood into arterial.
Venous blood, due to the fact that the hemoglobin it contains is converted into oxyhemoglobin, releases about 6 vol.% CO2 in the capillaries of the lungs. If hemoglobin in the lungs were not converted into oxyhemoglobin, then, as can be seen from the curve, venous blood with a partial pressure of carbon dioxide in the alveoli equal to 40 mm Hg. Art.. would bind 54 vol.% CO2, therefore, would give up not 6, but only 4 vol.%. Likewise, if the arterial blood in the capillaries of the systemic circle did not give up its oxygen, i.e., if its hemoglobin remained saturated with oxygen, then this arterial blood, at the partial pressure of carbon dioxide present in the capillaries of the body tissues, would not be able to bind 58 vol. .% CO2, but only 55 vol.%.
CARBONAN HYDRASE (carbonate dehydratase, carbonate hydrolyase, obsolete name - carbonic anhydrase; EC 4.2.1.1) - an enzyme that catalyzes the reversible reaction of the splitting of carbonic acid to carbon dioxide and water; is one of the most common and most active enzymes of the human body, is involved in such body functions as CO 2 transport, the formation of hydrochloric acid in the stomach and maintaining acid-base balance. The amount of K activity in human blood serves as a diagnostic test for a number of diseases.
Carbon dioxide, formed during tissue respiration in tissue capillaries, under the influence of red blood cells transforms into H 2 CO 3 (H + + HCO 3 -); H + ions are bound by hemoglobin (see), and HCO 3 - ions in the form of bicarbonate are transported with the blood to the lungs. In the pulmonary capillaries, under the influence of carbon dioxide, carbon dioxide is released from H 2 CO 3 and then removed from the body. K. kidneys participate in the process of water reabsorption in the renal tubules. A decrease in its catalytic activity leads to urine alkalosis (i.e., an increase in its pH values) and polyuria. K., ensuring the maintenance of acid-base balance, has a significant effect on the excitability and conductivity of nervous tissue. K. also catalyzes the hydrolysis of a number of esters and the hydration of aldehydes. The enzyme belongs to the class of lyases, a subclass of carbon-oxygen lyases.
K. was first discovered in erythrocytes by N. Meldrum and F. J. Boughton in 1932. K.’s activity is determined, in addition to erythrocytes, in the parietal cells of the gastric mucosa, in the cells of the adrenal cortex and kidneys, as well as in the cells of c. n. pp., pancreas, in the retina and lens of the eye and some other human organs.
K. mammals is a metalloenzyme (zinc protein).
There is 1 g-atom of zinc per 1 mole of enzyme protein; Zn 2+ can be replaced by Co 2+ without changing enzyme activity. Mn 2+, Fe 2+ and Ni 2+ ions are much less active in this regard.
Plant cells differ in their properties from cells isolated from animal and human tissues.
K. human erythrocytes have three isoenzymes (see) - A, B and C, of which the latter is distinguished by the highest activity. The ratio of these isoenzymes varies in different pathol states (normally it is 5%, 83% and 12%, respectively).
K. is inhibited by most monovalent anions, cyanide, sulfides, azides, phenols, and acetonitrile. Some sulfonamides and their derivatives are strong inhibitors of K. in animals and microorganisms, for example, acetazolamide - diacarb (see), which is used in medicine as a diuretic and anticonvulsant, as well as in the treatment of glaucoma.
K.'s activity in the blood of healthy people is quite constant, but in some pathol states it changes sharply. So, for example, with anemia of various etiologies, the specific activity of blood K increases; it also increases with circulatory disorders of the 2nd - 3rd degree, as well as with some lung lesions (bronchiectasis, pneumosclerosis). With intravascular hemolysis, K.'s activity is determined in the urine, where normally it is absent* In patients with low acidity of gastric juice, low K.'s activity in the blood is noted, and with increased acidity, K.'s activity in the blood is slightly increased.
Taking into account the widespread use in the clinic of Pharmakol, drugs that are inhibitors of K. (hypothiazide, diacarb, etc.), the advisability of systematic monitoring of K.’s activity in the blood of patients taking such drugs is obvious.
K.'s activity in wedges and laboratories is determined using the Brinkman method (see Brinkman method) modified by E. M. Kreps and E. Yu. Chenykaeva, as well as by the micro-method of A. A. Pokrovsky and V. A. Tutelyan, based by measuring the time required for the pH shift from 9.0 to 6.3 as a result of CO 2 hydration under the influence of K. of the blood sample under study. Normally, K activity, determined by this method, is 2.01 ± 0.08 units, and in terms of 1 million red blood cells, 0.458 ± 0.006 units. (for 1 unit of K activity, the acceleration of a catalyzed reaction is taken to be 2 times compared to a non-catalyzed one under standard conditions: temperature 0-1°, time 100-110 seconds, blood dilution 1: 1000).
Bibliography Crepe E. M. Respiratory enzyme - carbonic anhydrase and its significance in physiology and pathology, Usp. modern, biol., t. 17, v. 2, p. 125, 1944; L e-ninger A. Biochemistry, trans. from English, p. 177, M., 1974; L i n d s k o g S. a. o. Carbonic anhydrase, in: Enzymes, ed. by P. D. Boyer, v. 5, p. 587, N. Y.-L., 1971, bibliogr.; Scrutton M. Assay of enzymes of carbon dioxide metabolism, in the book: Meth. microbiol., ed. by J. R. Norris a. D. W. Ribbons, v. 6A, p. 479, L.-N. Y., 1971.
G. A. Kochetov.
Which, paradoxically, are not independently used as diuretics (diuretics). Carbonic anhydrase inhibitors are mainly used for glaucoma.
Carbonic anhydrase in the epithelium of the proximal tubules of the nephron catalyzes the dehydration of carbonic acid, which is a key link in the reabsorption of bicarbonates. When carbonic anhydrase inhibitors act, sodium bicarbonate is not reabsorbed, but is excreted in the urine (urine becomes alkaline). Following sodium, potassium and water are excreted from the body in the urine. The diuretic effect of substances in this group is weak, since almost all of the sodium released into the urine in the proximal tubules is retained in the distal parts of the nephron. That's why Carbonic anhydrase inhibitors are currently not used independently as diuretics..
Carbonic anhydrase inhibitor drugs
Acetazolamide
(diacarb) is the most famous representative of this group of diuretics. It is well absorbed from the gastrointestinal tract and, unchanged, is quickly excreted in the urine (that is, its effect is short-term). Drugs similar to acetazolamide - dichlorphenamide(daranid) and methazolamide(neptazane).
Methazolamide also belongs to the class of carbonic anhydrase inhibitors. Has a longer half-life than acetazolamide and is less nephrotoxic.
Dorzolamide. Indicated for the reduction of elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension who are insufficiently responsive to beta-blockers.
Brinzolamide(trade names Azopt, Alcon Laboratories, Inc, Befardin Fardi MEDICALS) also belongs to the class of carbonic anhydrase inhibitors. Used to reduce intraocular pressure in patients with open-angle glaucoma or ocular hypertension. The combination of brinzolamide and timolol is actively used on the market under the trade name Azarga.
Side effects
Carbonic anhydrase inhibitors have the following main side effects:
- hypokalemia;
- hyperchloremic metabolic acidosis;
- phosphaturia;
- hypercalciuria with risk of kidney stones;
- neurotoxicity (paresthesia and drowsiness);
- allergic reactions.
Contraindications
Acetazolamide, like other carbonic anhydrase inhibitors, is contraindicated in cirrhosis of the liver, since alkalinization of the urine prevents the release of ammonia, which leads to encephalopathy.
Indications for use
Carbonic anhydrase inhibitors are primarily used to treat glaucoma. They can also be used to treat epilepsy and acute mountain sickness. Since they promote the dissolution and elimination of uric acid, they can be used in the treatment of gout.
Acetazolamide used in the following conditions:
- Glaucoma (reduces the production of intraocular fluid by the choroid plexus of the ciliary body.
- Treatment of epilepsy (petit mal). Acetazolamide is effective in treating most types of seizures, including tonic-clonic and absence seizures, although it has limited benefit as tolerance develops with long-term use.
- For the prevention of nephropathy during treatment, since the breakdown of cells releases a large amount of purine bases, which provide a sharp increase in the synthesis of uric acid. Alkalinization of urine with acetazolamide due to the release of bicarbonates inhibits nephropathy due to the loss of uric acid crystals.
- To increase diuresis during edema and correct metabolic hypochloremic alkalosis in CHF. By reducing the reabsorption of NaCl and bicarbonates in the proximal tubules.
However, for none of these indications is acetazolamide the primary pharmacological treatment (drug of choice). Acetazolamide is also prescribed for mountain sickness (as it causes acidosis, which leads to the restoration of the sensitivity of the respiratory center to hypoxia).
Carbonic anhydrase inhibitors in the treatment of mountain sickness
At high altitudes, the partial pressure of oxygen is lower, and people must breathe faster to get enough oxygen to live. When this happens, the partial pressure of carbon dioxide CO2 in the lungs is reduced (simply blown out when you exhale), resulting in respiratory alkalosis. This process is usually compensated by the kidneys through bicarbonate excretion and thereby causes compensatory metabolic acidosis, but this mechanism takes several days.
More immediate treatment is carbonic anhydrase inhibitors, which prevent bicarbonate uptake in the kidneys and help correct alkalosis. Carbonic anhydrase inhibitors also improve chronic mountain sickness.
