home Ceiling The meaning of the van den Bergh reaction in medical terms. Chemical tests to detect bile pigments

The meaning of the van den Bergh reaction in medical terms. Chemical tests to detect bile pigments

Bilirubin(lat. bilis bile + ruber red) is one of the yellow-red bile pigments.

Chemical composition bilirubin molecules - C 33 H 36 O 6 N 4. Molecular weight - 584.68. In its pure form, bilirubin is a crystalline substance consisting of rhomboidal-prismatic crystals of yellow-orange or red-brown color, sparingly soluble in water.

The bilirubin molecule, like all its derivatives, is based on four pyrrole rings inherited from hemoglobin. Two hydroxyl groups determine the acidic chemical properties of bilirubin and its ability to form salts. The arrangement of hydroxyl groups has variations, the main of which is their attachment to 2 and 3 pyrrole rings (bilirubin IXα).

Chemical forms of bilirubin and their names

Traditionally, there are two main chemical forms bilirubin:

free bilirubin + glucuronic acid = bound bilirubin

In this case, bilirubin can be associated with one molecule of glucuronic acid (bilirubin monoglucuronide) or two (bilirubin biglucuronide).

Bilirubin is also divided into indirect And straight. It has long been the practice that indirect bilirubin is identified with free bilirubin, and direct bilirubin with bound bilirubin. But this is not entirely true.

About the difference between direct and bound bilirubin

The fact is that the terms “indirect” and “direct” bilirubin are not chemical, but technological and reflect the result of bilirubin widely used for laboratory identification. Since it was believed that indirect bilirubin was free, and direct bilirubin was exclusively bound, these terms were used interchangeably.

This simple circuit lost its correctness after another chemical form of bilirubin was discovered: biliprotein, or delta bilirubin.

Delta-bilirubin takes part in the Van den Bergh reaction similarly to bound bilirubin and is taken into account together with it as direct bilirubin. Therefore the formula "direct bilirubin = conjugated bilirubin" is true only for healthy people in whom delta-bilirubin is practically absent. In some painful conditions, direct bilirubin can consist of 60-90% delta-bilirubin:

direct bilirubin = conjugated bilirubin + delta bilirubin

Read about the properties and diagnostic value below.

Sources of bilirubin formation

Almost the only source of bilirubin in the body is heme.

Heme is a structure that includes, like bilirubin, four pyrrole rings, and in addition, an iron atom. Heme is part of the molecules hemoglobin(red blood cell oxygen-carrying protein), muscle contractile protein myoglobin and cellular enzymes cytochromes.

The main erythrocyte flow of bilirubin (85% of the total amount) is formed in the process of recycling hemoglobin from obsolete (about 120 days) red blood cells. Such red blood cells are removed from the bloodstream and destroyed mainly in the spleen, but also in the liver and bone marrow. An increase in bilirubin of erythrocyte origin occurs during hemolysis (the so-called massive breakdown of red blood cells).

Other sources give rise to the so-called. shunt bilirubin, which accounts for up to 15% of its total amount. Among the sources of shunt bilirubin are:

Defective red blood cells and their immature precursors. As a result of constant rejection, such cells die soon after their birth, not having time to leave the “incubator” of blood cells - the bone marrow. The number of such cells, and accordingly, the amount of shunt bilirubin increases sharply in some hereditary, autoimmune, and tumor diseases of the blood system. Under normal conditions, bone marrow produces no more than 7% bilirubin.
A small amount of bilirubin is formed during the process of constant renewal and destruction of the muscle protein myoglobin. However, injuries that involve extensive destruction of muscle tissue can lead to a short-term increase in bilirubin levels in the blood.
A small portion of bilirubin also comes from all tissues of the body as a result of the breakdown of cytochromes and some proteins that do not contain heme, in particular peroxidases.

Densitometric studies using radiolabeled glycine have shown that the heme that gives rise to shunt bilirubin lasts no more than 10 days. At the same time, the lifespan of heme in normal erythrocytes coincides with the lifespan of the erythrocytes themselves - 120 days. Knowing the proportion of shunt bilirubin in its total concentration, one can judge the nature of the disease process, but so far such an analysis is only available in scientific studies.

The transformation of heme into bilirubin, among many other tasks, is occupied tissue macrophages, which are part immune system body. Tissue macrophages are present in all organs, usually located in their connective tissue, but they are predominantly concentrated in the spleen, lymph nodes, liver and bone marrow. All tissue macrophages trace their ancestry to monocytes grown in the hematopoietic matrix of the bone marrow, but in various organs they are specialized to perform special tasks, and therefore have special names - for example, Kupffer cells of the liver, histiocytes of the spleen, etc. Previously, the system of tissue macrophages was called reticuloendothelial system, but this term is now considered obsolete.

80% of bilirubin is produced by Kupffer cells of the liver
the remaining part is macrophages of the bone marrow and spleen
a very small amount - histiocytes of the connective tissue of all organs

Every day, 2*10 8 aged red blood cells are destroyed in the human body. This releases 6-8 g of hemoglobin, from which, in turn, 250-350 mcg of bilirubin originates.

The released heme is absorbed by macrophages, after which it is converted with the participation of the intracellular enzyme heme oxygenase into an intermediate substance biliverdin. At the same time, an iron atom and carbon monoxide are split off from the heme. Biliverdin, having a green color, belongs to the group of pigment substances. In addition, unlike bilirubin, it is highly soluble in water.

At the second stage of heme cleavage using the enzyme biliverdin transferase, biliverdin is transformed into bilirubin, a yellow-orange pigment substance insoluble in water. As you know, each hemoglobin molecule contains four heme units. Accordingly, when one hemoglobin molecule is broken down, four bilirubin molecules and four iron atoms are formed.

As already mentioned, carbon monoxide (CO) is released as a byproduct when the carbon bonds of heme are broken down, and this is the only reaction in the body that produces this substance. This circumstance is used in a promising research method: by measuring the concentration of carbon monoxide in exhaled air, it is possible to estimate the rate of breakdown of heme in the body.

The sequential conversion of heme into green biliverdin, and then into yellow-red bilirubin explains the change in color of bruises after contusions from dark blue to blue-green, and then to yellow.

Initially, only free bilirubin is formed in the body. Bound bilirubin appears later as a result of the transformation of free bilirubin.

Properties of free bilirubin

So, in our body there is a constantly operating conveyor belt for the production of free bilirubin as part of the physiological mechanism of replacing old red blood cells. This gives rise to several difficult problems to solve:

Free bilirubin is a toxic substance, and therefore a reliable mechanism is needed to remove it from the body and maintain its concentration at a safely low level. Bilirubin toxicity occurs primarily in brain tissue. Even moderately increased level free bilirubin is manifested by unexpressed symptoms from the nervous system: loss of attention, fatigue, etc. However, in adults, the concentration of free bilirubin never reaches such a dangerous level as to cause damage to the nervous system. But this happens in newborns. In particular, an immunological conflict can lead to massive hemolysis and the development of the so-called. "nuclear" jaundice. In this case, free bilirubin, the level of which in the blood serum can increase tens of times and reach 300 µmol/l and higher, accumulates in the brain tissue, causing irreversible changes in the subcortical nuclei of the brain.
Free bilirubin is practically insoluble in water. Since all biological fluids of the body are aqueous solutions, free bilirubin cannot be removed from the body in the form in which it is without changing its chemical structure.
At the same time, free bilirubin is highly soluble in fats. Thanks to this property, it easily overcomes the so-called phospholipid membranes. the blood-brain barrier, designed to protect the brain tissue from the penetration of numerous toxic substances circulating in the bloodstream.

In the bloodstream, free bilirubin is transported by albumin proteins, holding it on its surface. 1g of albumin carries 8.5mg of bilirubin. The albumin-bilirubin complex is fragile and disintegrates as soon as possible.

The Van den Bergh reaction identifies free bilirubin as indirect.

Bilirubin as an antioxidant

In the light of the latest research, the traditional idea of bilirubin as a uniquely “slag” product has changed somewhat. Free bilirubin has pronounced antioxidant properties, and the body actively uses them.

The antioxidant activity of bilirubin significantly exceeds that of α-tocopherol (vitamin E), which is considered a classic antioxidant. Thus, bilirubin inactivates H 2 O 2 in a concentration that is 10 times higher than its own. In particular, it prevents lipid peroxidation of cell membranes, as well as the oxidation of membrane proteins. The most significant antioxidant properties of bilirubin are in relation to nervous tissue and cardiac muscle, since these tissues do not have a sufficiently powerful own defense against free radicals.

It was found that people with chronically high levels of free bilirubin are significantly less likely to suffer from vascular atherosclerosis and related heart diseases. An inverse relationship between the level of free bilirubin in the blood and cardiac pathology has been proven.

One study of 10,000 patients showed lower mortality from tumor diseases among people with high levels of free bilirubin.

In light of these data, it seems appropriate to transform the highly water-soluble biliverdin into the “inconvenient” insoluble bilirubin.

Conversion of free bilirubin into bound

Since, as mentioned above, free bilirubin is insoluble in water, its removal from the body is impossible without first transforming it into other water-soluble substances.

The specific site of such transformation is the main structural unit of the liver - the liver cell, or hepatocyte. Hepatocytes are collected in liver lobules. The hepatic lobule is designed in such a way that each liver cell, on one side, has contact with a venous blood capillary (the so-called sinusoid), and on the other, a bile capillary is connected to it.

Free bilirubin, transported on the surface of albumin, from the venous blood of the sinusoid moves first into the space of Disse, separating the capillary and the liver cell, and then through the cell membrane into the hepatocyte, simultaneously freeing itself from binding with albumin. This movement occurs without energy consumption due to the concentration difference.

Inside the cell, bilirubin binds reversibly to ligandin proteins. Ligandins prevent the “escape” of bilirubin back into the venous capillary, and also transport it to the mesh structure - the endoplasmic reticulum.

In the endoplasmic reticulum there are so-called. microsomes are vesicles filled with enzymes. On the surface of microsomes, with the catalytic participation of the enzyme glucuronyltransferase, a reaction occurs between free bilirubin and glucuronic acid, resulting in the appearance of a new substance - bound or conjugated bilirubin.

This reaction can take place in one or two cycles, yielding bilirubin monoglucuronide or biglucuronide, respectively. The ratio of monoglucuronide to biglucuronide is 4:1.

Gluuronidation of bilirubin is one of the bottlenecks in its metabolism, since it is limited by the amount of glucuronyl transferase enzyme. This amount is sharply reduced (less than 1-3% of the norm) in some hereditary diseases, in particular with.

Difficult release of conjugated bilirubin into bile leads to its accumulation in the blood. In such cases, the kidneys are forced to take on the function of removing it from the body, although under normal conditions they do not do this. The appearance of bilirubin in the urine is a sign of a serious illness.

Properties of bound bilirubin

Bound bilirubin differs favorably from free bilirubin in its properties:

it is non-toxic
highly soluble in aqueous media
It is easily excreted from the body, mainly with bile, and, if necessary, with urine.

Bound bilirubin takes part in the direct van den Bergh reaction and is therefore usually called direct bilirubin.

It should also be noted that conjugated bilirubin has a bad property: when it is excessively concentrated in bile, it is prone to crystallization and the formation of bilirubin stones in the bile. gallbladder. Since a high concentration of conjugated bilirubin is a consequence of increased formation of free bilirubin in the body, the causes of such conditions are usually other diseases of the blood system.

On the decisive role of the liver in the metabolism of bilirubin

Thus, the only body in human body The liver is capable of converting free bilirubin into bound bilirubin.

The crucial role of the liver in the metabolism of bilirubin is even more obvious when we consider that 80% of free bilirubin is also produced in the liver by Kupffer cells. The level of bilirubin is one of the most reliable indicators of the functional state of the liver, since almost the entire process of bilirubin metabolism is closely dependent on this function.

It is no coincidence that the responsibility for the disposal of bilirubin is assigned to the liver, which is rightly called the main chemical laboratory of the body. The liver converts a huge number of substances into non-toxic chemical forms, both naturally formed in the body and entering it from the outside, including medicinal substances.

Some metabolic end products are primarily excreted from the body through the kidneys with urine, others through the liver with bile. Which of the two pathways is preferable for each specific substance is determined by the peculiarities of its chemical structure and the physiology of the liver and kidneys. General principle is as follows: the kidneys are good at excreting substances with a molecular weight of less than 300 c.u. That is, the rest are excreted mainly in bile, including bilirubin.

It should be said that to process bilirubin, the liver cell uses universal enzyme systems, which, along with bilirubin, take part in the metabolism of many other substances. Along with saving the body's resources, this situation sometimes leads to negative consequences. The fact is that a number of substances, in particular many medications compete with bilirubin in enzymatic reactions and, in case of overdose, are able to completely remove the latter from the metabolic process. This leads to the accumulation of indirect bilirubin in the body and to the development of the so-called. "unconjugated" jaundice. These drugs include paracetamol and some other non-steroidal analgesics, some antibiotics.

Bound bilirubin is released from the hepatocyte into the bile capillary and excreted with bile into the intestine. The release of bound bilirubin into the bile capillary requires energy, so damage to liver cells during hepatitis, cirrhosis, etc. leads to disruption of this process.

Transformation of bilirubin in the intestine and its products

Thus, bound bilirubin is released with the flow of bile into the intestine. It is bilirubin that gives bile its dirty green hue.

Since microorganisms living in the intestine are actively working on its contents, bilirubin also undergoes further transformation. During the processing of bilirubin in the intestines, numerous intermediate substances are formed. The process of processing bilirubin in the intestine is multi-stage and occurs with the formation of numerous intermediate substances.

The main stages of intestinal transformation:

Under the influence of the bacterial enzyme β-glucuronidase, conjugated bilirubin undergoes hydrosis (cleavage) to form free bilirubin
Free bilirubin, as a result of a series of reduction reactions, is transformed into a number of substances under the general name “urobilinogens” or “urobilinoids”. Urobilinogens, like bilirubin, are based on a structure of four pyrrole rings. Urobilinogens are colorless.

Among the urobilinogens, the most important substances are:

mesobilinogen - the ancestor of the urobilinogen group
stercobilinogen
urobilinogen itself

Most of the urobilinogens are eventually transformed into the final pigment products - stercobilin and urobilin, which are orange-brown in color. It is these substances that give feces their characteristic color. Discoloration of feces indicates a lack of bilirubin in them, which happens with hepatitis or when the bile ducts are blocked. 10-20% of urobilinogens are absorbed from the intestine and returned to the liver through the portal vein system. The liver does what it knows how to do with them: it converts it into bound bilirubin and sends it back to the intestines. Normally, no more than 2-5% of urobilinogens are excreted in the urine. Such a small amount is not detected by routine laboratory urine testing.

With hepatitis, the liver cannot cope with the utilization of urobilinogens, as a result of which they are found in the urine. - an important diagnostic sign of liver diseases.

Van den Berg reaction and other methods for determining bilirubin

It is time to explain the origin of the somewhat strange names: “direct” and “indirect” bilirubin. And the two forms of bilirubin were named this way thanks to the light hand of Mr. Van Den Berg, who developed a reaction for identifying bilirubin in blood serum using Ehrlich’s reagent back in 1916. After 100 years, the reaction, named the Van den Bergh reaction in his honor, remains the main method in laboratory practice for studying bilirubin content.

Without delving into the details of the technique, which has undergone numerous modifications over a hundred years, we note its fundamental feature - a two-stage implementation:

First stage (direct Van den Bergh reaction): Ehrlich's reagent is added to the test tube with the blood serum being tested. Soon, if there is bound bilirubin in the serum, the contents of the test tube acquire a bright pink color. Free bilirubin, blocked by albumin, does not take part in the reaction. Using a colorimetric device, the quantitative content of bound bilirubin can be determined by the color intensity. Thus, the bound bilirubin that takes part in the direct reaction is called “direct” bilirubin.
Second phase (indirect Van den Bergh reaction): a substance that precipitates albumins is first added to another test tube with the test serum. In Van den Berg's original version, 96˚ ethyl alcohol was used for this purpose. Subsequently, ethyl alcohol was replaced with more effective substances. At the same time, albumin settles to the bottom of the test tube, and unblocked free bilirubin acquires the ability to enter into a chemical reaction with Ehrlich’s reagent, which it does. Simultaneously with indirect bilirubin, direct bilirubin (if any) also participates in the reaction, that is, the indirect Van den Bergh reaction determines total bilirubin as the sum of direct and indirect bilirubin. The content of indirect bilirubin is calculated as the difference between total and direct bilirubin:
indirect bilirubin = total bilirubin - direct bilirubin

The Van den Bergh reaction, along with its undoubted advantages, including ease of implementation and clarity of the result, also has a significant drawback - it produces an overestimated level of direct bilirubin. Thus, in the serum of healthy people, this method detects up to 5.4 µmol/l of direct bilirubin, which accounts for up to 25% of the total. In fact, as more accurate methods show, in these people the total bilirubin is almost 100% indirect, and direct bilirubin is practically absent. However, in clinical practice, it is more important to know not the content of bilirubin itself, but its dynamics, for which it is necessary to ensure the comparability of results obtained at different times and in different laboratories.

α - free bilirubin
β - bilirubin monoglucuronide
λ - bilirubin biglucuronide

Other methods for detecting bilirubin have been developed, detailed description which are beyond the scope of this article:

spectrophotometry
gas analyzer
high performance liquid chromatography
non-invasive method - reflective densitometry

Among these methods, high-performance liquid chromatography is the most promising. This technique allows you to determine the content of four fractions of bilirubin:

α - free bilirubin
β - bilirubin monoglucuronide
λ - bilirubin biglucuronide
δ - delta-bilirubin, or biliprotein

Delta bilirubin

Delta bilirubin is a compound of conjugated bilirubin (biglucuronide or bilirubin monoglucuronide) with albumin. In the scientific literature, another name for this substance is often used: “biliprotein”. This substance is yellow in color.

The Van den Bergh reaction does not allow delta-bilirubin to be determined separately and identifies it together with bilirubin glucuronides as direct, since all these substances react directly with Ehrlich's reagent in a similar way. To selectively determine its content in blood serum, high-performance liquid chromatography is used.

In the biochemical and physiological aspects, delta-bilirubin has a number of features that generally allow us to consider it, along with free and bound bilirubin, as an independent, third form of bilirubin:

Unlike the complex of albumin with free bilirubin, delta-bilirubin is a fairly stable substance, since its components are irreversibly linked by a strong covalent chemical bond.
The synthesis of the delta-bilirubin molecule does not require the participation of intracellular elements, therefore it is possible in vivo and in vitro, i.e. both in the body and in vitro.
Delta-bilirubin is non-toxic to body tissues.
Due to the excessive massiveness of its molecule, delta-bilirubin is not excreted unchanged from the body either with bile or through the kidneys.
It has been established that the half-life of delta-bilirubin in the body is the same as that of ordinary albumin and is 14-21 days. Only after the destruction of albumin does it become possible to remove the bilirubin glucuronide previously combined with it from the body, mainly with bile.

Under normal conditions, delta-bilirubin is not detected in significant quantities. Its content increases sharply with cholestasis, i.e., a violation of the production and secretion of bile components against the background of preserved function of the synthesis of conjugated bilirubin. Cholestasis can be either intrahepatic in hepatitis and cirrhosis of the liver, or extrahepatic, caused by difficulty in the outflow of bile in the extrahepatic bile ducts, which usually happens when they are blocked by a gallstone.

The content of delta-bilirubin in cholestasis can reach 60-70 and even 90% of direct bilirubin. Due to its “survivability” in the body, delta-bilirubin (and at the same time direct bilirubin in general) remains high for another 1-1.5 weeks after the outflow of bile is normalized. This explains the previously incomprehensible phenomenon when direct bilirubin long time remains high, despite obvious clinical improvement and normalization of other laboratory parameters, in particular.

At the same time, there is no increase in the delta-bilirubin content with unconjugated hyperbilirubinemia (accumulation of unconjugated bilirubin in the blood, which occurs with and some other conditions).

Lumirubin and other bilirubin photoproducts

In its usual and most stable configuration, the bilirubin molecule is folded in such a way that some active groups are blocked by others, which determines its insolubility. Under the influence of blue light, bilirubin can be converted into numerous photoproducts, most of which are highly soluble in water due to unblocked hydroxyl groups

At one time, chemists became interested in the paradoxical solubility of free bilirubin. Theoretically, the bilirubin molecule, due to the presence of two hydroxyl groups COOH, should have pronounced polarity. As is known, substances with a polarized molecule are soluble in water and insoluble in fats. In reality, free bilirubin behaves in the opposite way.

The mystery was solved using X-ray crystallography. It turned out that the free bilirubin molecule has a spatial configuration in which the polarizing hydroxyl groups are blocked by internal hydrogen bonds (so-called Z-Z connection). This configuration of the bilirubin molecule is the most stable and is the main one, since it has a minimum of space and energy. It has also been discovered that there are numerous other variations along with the basic configuration.

Of greatest interest among them are photo products bilirubin, which are formed under the influence of blue light in the presence of atomic oxygen. Bilirubin molecules, absorbing the energy of light photons, change internal interatomic Z-Z bonds to higher-energy Z-E and E-E bonds. At the same time, along with a change in the spatial configuration of the molecules, their properties also radically change - they become water-soluble.

Due to their good water solubility, photoproducts are quickly eliminated from the body by the liver. One of the photoproducts with the highest elimination rate is E-E connections, named lumirubine. Photoproducts have a short lifespan because such molecules get rid of excess energy as soon as possible and return to their original, basic configuration.

The ability of free bilirubin to form water-soluble photoproducts is used to treat neonatal jaundice using phototherapy.

Read the continuation:

In clinical practice, various methods for determining bilirubin and its fractions in blood serum are used.

The most common of these is biochemical Jendrassik-Grof method

It is based on the interaction of bilirubin with diazotized sulfanilic acid to form azopigments. In this case, bound bilirubin (bilirubin-glucuronide) gives a fast (“direct”) reaction with the diazo reagent, while the reaction of free (not bound to glucuronide) bilirubin proceeds much more slowly. To accelerate it, various accelerator substances are used, for example caffeine (Jendrassik-Cleghorn-Grof method), which release bilirubin from protein complexes (an “indirect” reaction). As a result of interaction with diazotized sulfanilic acid, bilirubin forms colored compounds. Measurements are carried out using a photometer.

PROGRESS OF DETERMINATION

Reagents are introduced into 3 test tubes (2 experimental samples and a blank) as indicated in the table. Diazoreaction

To determine bound bilirubin, the measurement is carried out 5-10 minutes after adding the diazo mixture, since during prolonged standing, unbound bilirubin reacts. To determine total bilirubin, the color development sample is left to stand for 20 minutes, after which it is measured on a photometer. The color does not change upon further standing. The measurement is carried out at a wavelength of 500-560 nm (green filter) in a cuvette with a layer thickness of 0.5 cm against the water. The blank sample is subtracted from the values obtained by measuring total and conjugated bilirubin. The calculation is made according to the calibration schedule. The content of total and bound bilirubin is determined. The method of Jendrassik, Cleghorn and Grof is simple, convenient in practice, does not involve the use of scarce reagents, and is the most acceptable for practical laboratories. It is recommended to carry out the determination immediately after sampling to avoid oxidation of bilirubin in the light. Serum hemolysis reduces the amount of bilirubin in proportion to the presence of hemoglobin. Therefore, blood serum should not be hemolyzed.

A number of substances - hydrocortisone, androgens, erythromycin, glucocorticoids, phenobarbital, ascorbic acid - cause interference.

Construction of the calibration graph using the Jendrassik method.

Method I - Shelonga-Vendes using the stabilizing properties of blood serum protein. Stock bilirubin solution: in a 50 ml flask, dissolve 40 mg of bilirubin in 30-35 ml of 0.1 mol/l sodium carbonate solution Na2CO3. Shake well, avoiding the formation of bubbles. Adjust to 50 ml with 0.1 mol/l Na2CO3 solution and stir several times. The solution is stable only for 10 minutes from the start of preparation. Subsequently, bilirubin is oxidized. Bilirubin working solution: to 13.9 ml of fresh non-hemolyzed serum of a healthy person add 2 ml of freshly prepared bilirubin stock solution and 0.1 ml of 4 mol/l acetic acid solution. Mix well. This releases bubbles of carbon dioxide. The working solution is stable for several days. This solution contains exactly 100 mg/L, or 171 µmol/L, more bilirubin than the serum taken to prepare the solution. To exclude the amount of bilirubin contained in this serum from the calculations, when measuring on a photometer, the extinction values of the corresponding dilutions of the compensation liquid are subtracted from the extinction values of the calibration samples. To prepare the compensation fluid, mix 13.9 ml of the same serum that was used to prepare the bilirubin calibration solution, 2 ml of a 0.1 mol/L sodium carbonate solution and 0.1 ml of a 4 mol/L acetic acid solution. To construct a calibration curve, a series of dilutions with different bilirubin contents are prepared. To the resulting dilutions add 1.75 ml of caffeine reagent and 0.25 ml of diazo mixture. If cloudiness appears, you can add 3 drops of a 30% sodium hydroxide solution. The measurement is carried out under the same conditions as in the experimental samples, after 20 minutes. Dilutions similar to the calibration ones are prepared from the compensation liquid (as indicated below), and then they are processed in the same way as calibration samples.

To determine bilirubin and its fractions in blood plasma, various methods are used, the most common of which is the Jendrasik-Grof method. The method is based on the interaction of bilirubin with sulfanilic acid and sodium nitrate. At the same time, bound bilirubin gives a fast reaction, which is why it was given the name direct, and free bilirubin gives a slower reaction, to accelerate which various substances are used (caffeine, ethanol, etc., which first release bilirubin from protein complexes), which is why it is called indirect.

Various methods for detecting bilirubin in urine are based on its transformation under the influence of oxidizing agents into other substances that have a green or red color.

For determining bilirubin a colorimetric method is used, which is based on Van den Bergh reaction.

The reaction proceeds in 2 stages: in the first, under the influence of hydrochloric acid, the tetrapyrrole chain is broken, resulting in the formation of two dipyrroles, in the second, both dipyrrolic derivatives are diazotized by diazophenylsulfonic acid, converting them into azobilirubin.

Fractional bilirubin content is determined using the modified Van den Bergh method proposed by Jendrassik.

The method is accepted as unified.

The formation of bilirubin is based on the degradation of heme in hemoglobin and other heme-containing proteins and enzymes. Heme breaks down to biliverdin, which is reduced to bilirubin.

Free bilirubin is toxic, insoluble in water and circulates in the blood in combination with albumin. This bilirubin gives an indirect Van den Berg reaction (after precipitation of albumin with alcohol), and is therefore called indirect.

Indirect bilirubin, being bound to albumin, does not pass through the intact glomerular membranes and is not filtered into the urine.

Bilirubin is excreted with bile through the intestines. Bilirubin bound to albumin is carried by the blood to the liver. Bilirubin easily penetrates the membranes of hepatocytes, albumin remains in the bloodstream. In hepatocytes, bilirubin undergoes conjugation with glucuronic acid, turning into bilirubin mono- and diglucuronide. The resulting bilirubin glucuronides are non-toxic and easily soluble. They are sent with bile to the intestines for excretion from the body.

From the intestine, bilirubin glucuronides partially enter the bloodstream and, while in the blood, represent a fraction of direct bilirubin, which gives a direct Van den Bergh reaction.
Direct bilirubin, unlike indirect bilirubin, easily penetrates the kidney filters and can be excreted in the urine.

Under physiological conditions, blood serum contains approximately 25% direct bilirubin (bound to glucuronic acid) and 75% indirect bilirubin (albumin-bilirubin).

Thus, total blood bilirubin is the total amount of indirect and direct bilirubin.

In healthy people, the blood serum contains bilirubin 1.7-20.5 µmol/l; direct - 0.4-5.1 µmol/l.

To determine bilirubin, a colorimetric method is used, which is based on the Van den Bergh reaction. The reaction proceeds in two stages: in the first, under the influence of hydrochloric acid, the tetrapyrrole chain is broken and two dipyrroles are formed, in the second, both dipyrrolic derivatives are diazotized by diazophenylsulfonic acid, converting them into azobilirubin. Fractional bilirubin content is determined using the modified Van den Bergh method proposed by Jendrassik.
The method is accepted as unified.

Principle of the method

Bilirubin reacts with azo coupling with diazotized sulfanilic acid to form an azo dye solution. The color intensity is proportional to the bilirubin content and is detected photometrically.

The original Van den Bergh method establishes only the nature of the reaction (direct, indirect, slow), i.e. There is a predominance of one or another bilirubin in the blood serum. The method of Jendrassik, Cleghorn and Grof makes it possible to fractionally determine the bilirubin content. It is simple, easy to use, does not involve the use of scarce reagents, and is most suitable for practical laboratories.

It is recommended to carry out the determination immediately after sampling to avoid oxidation of bilirubin in the light. Serum hemolysis reduces the amount of bilirubin in proportion to the presence of hemoglobin. Therefore, the blood serum should not be hemolyzed.

(A. A. H. Van den Bergh, 1869-1943, Danish doctor)
a method for the qualitative and quantitative determination of bilirubin in blood serum, based on the appearance of a red or pink color when it interacts with Ehrlich’s diazoreagent; quantitation produced colorimetrically.

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Adamkiewicz Reaction- (A. Adamkiewicz, 1850-1921, Austrian pathologist; synonym Adamkiewicz-Hopkins-Kohl reaction) color qualitative reaction to tryptophan and tryptophan-containing proteins, based on violet-blue........
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Adamkiewicz-Hopkins-Kohl Reaction- (A. Adamkiewicz, 1850-1921, Austrian pathologist; G. Hopkins, 1861-1947, English biochemist; L. Cole, born in 1903, French pathologist) see Adamkiewicz reaction.
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Adaptive Response— see Adaptive reaction.
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Allergic reaction- general name of clinical manifestations hypersensitivity body to the allergen.
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Delayed Allergic Reaction— (syn. kithergic reaction) A. r., developing within 24-48 hours after exposure to a specific allergen; in the occurrence of A. r. h. i.e. the main role belongs to......
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Allergic Reaction of Immediate Type- (syn. chimergic reaction) AR, developing after 15-20 minutes. after exposure to a specific allergen, e.g. with anaphylactic shock; in the occurrence of A. r. n. T.........
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Allergic Reaction Cross- A. r. to cross-reacting (common) antigens.
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Allergoid Reaction— (nrk) see Anaphylactoid reaction.
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Anamnestic Reaction- the body’s immune response to the repeated introduction of an antigen, characterized by a significantly higher titer of antibodies and a shorter period of their appearance compared......
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Anaphylactoid Reaction- (anaphylaxis + Greek eidos type; synonym: allergoid reaction nrk, anaphylatoxic reaction, phenomenon of parahypergia) - a nonspecific allergic reaction characterized by......
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Anaphylactic Reaction— see Anaphylactoid reaction.
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Antabuse-alcohol reaction— (syn. teturam-alcohol reaction) a complex of vegetative-somatic symptoms (skin hyperemia followed by pallor, tachycardia, shortness of breath, a sharp decrease in arterial......
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Aristovsky-Fanconi Reaction- (historical; V. M. Aristovsky, 1882-1950, Soviet microbiologist and immunologist; G. Fanconi, born in 1892, Swiss pediatrician) allergic intradermal test with a suspension of killed streptococci for...... ..
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Agglutination Reaction— (RA) is a method for identifying and quantifying Ag and Ab, based on their ability to form agglomerates visible to the naked eye. In the department of infectious diseases. diseases........
Dictionary of microbiology

Agglutination Reaction on Glass— - an express method for staging RA, in which the immune system and corpuscular Ag are mixed on the surface of a clean slide or special glass (with rings) In connection........
Dictionary of microbiology

Agglutination Inhibition Reaction— inhibition of Ag agglutination by homologous Abs as a result of preliminary contact of Abs with test Ags, usually of hapten nature. Based on competition of Ags for paratope Abs Highly sensitive
Dictionary of microbiology

Agglutination-Lysis Reaction- see Leptospirosis.
Dictionary of microbiology

Reaction- (slang) - here: a rapid fall in prices after a previous rise.
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VAN DEN BERG REACTION

(a.a.n. van den Bergh, 1869-1943, Danish doctor) a method for the qualitative and quantitative determination of bilirubin in blood serum, based on the appearance of a red or pink color when it interacts with Ehrlich’s diazoreagent; quantification is carried out colorimetrically.

Medical terms. 2012

The meaning of the van den Bergh reaction in medical terms. Chemical tests to detect bile pigments

Chemical forms of bilirubin and their names

About the difference between direct and bound bilirubin

Sources of bilirubin formation

Properties of free bilirubin

Bilirubin as an antioxidant

Conversion of free bilirubin into bound

Properties of bound bilirubin

On the decisive role of the liver in the metabolism of bilirubin

Transformation of bilirubin in the intestine and its products

Van den Berg reaction and other methods for determining bilirubin

Delta bilirubin

Lumirubin and other bilirubin photoproducts

View value Van den Bergh Reaction in other dictionaries

See also interpretations, synonyms, meanings of the word and what VAN DEN BERG REACTION is in Russian in dictionaries, encyclopedias and reference books:

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The meaning of the van den Bergh reaction in medical terms. Chemical tests to detect bile pigments

Chemical forms of bilirubin and their names

About the difference between direct and bound bilirubin

Sources of bilirubin formation

Properties of free bilirubin

Bilirubin as an antioxidant

Conversion of free bilirubin into bound

Properties of bound bilirubin

On the decisive role of the liver in the metabolism of bilirubin

Transformation of bilirubin in the intestine and its products

Van den Berg reaction and other methods for determining bilirubin

Delta bilirubin

Lumirubin and other bilirubin photoproducts

View value Van den Bergh Reaction in other dictionaries

See also interpretations, synonyms, meanings of the word and what VAN DEN BERG REACTION is in Russian in dictionaries, encyclopedias and reference books:

We recommend other articles

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