Hyaluronic acid (hyaluronate) is one of the most important components of extracellular tissue structures, a substance that is part of most biological fluids and performs a number of vital functions in the human body. In the body of a young man weighing 70 kg, about 15 g of this compound is present. Moreover, more than a third of its reserves undergo transformation (synthesis or breakdown) every day.

It has been proven that over time the concentration of hyaluronic acid in the body decreases. For example, in the organs and tissues of a person who has reached the age of 50, 30–40% less of this compound is present than in the body of a 17-year-old teenager. For this reason, modern nutritionists recommend that every person who has reached the age of 33–35 years increase the intake of this substance from the outside, that is, with food.

Hyaluronate was first isolated by scientists K. Meyer and D. Palmer from the vitreous body of a cow's eye in 1934. The chemical structure of this compound was determined much later - in the second half of the last century. As for the medical and biological properties of hyaluronic acid, their study is still ongoing.

Biological functions of hyaluronate

Hyaluronic acid is a vital substance for humans that performs a wide range of biochemical functions. To date, it has been proven that this compound:

• is the most important component of epithelial, connective and nervous tissues, biological fluids;
• increases the intensity of sodium, potassium, magnesium metabolism in cells;
• maintains optimal fluid balance in all tissues of the human body;
• prevents premature aging;
• accelerates regeneration processes by activating the secreting ability of fibroblasts (the cells that make up connective tissue);
• accelerates the processes of bone tissue fusion in case of fractures and other injuries;
• gives a viscous consistency to synovial fluid;
• creates optimal conditions for cell proliferation (division) and migration;
• improves blood microcirculation;
• increases the speed of transport of nutrients throughout the body;
• protects organs and tissues from injury due to compression;
• provides skin protection from the negative effects of direct sunlight;
• stimulates the processes responsible for the synthesis of elastin and collagen;
• has a pronounced anti-inflammatory effect;
• is one of the components that make up articular cartilage and ensures their normal functioning;
• eliminates the consequences of internal intoxication;
• protects the body from microbes (activates bactericidal factors on the wound surface and on the skin);
• changes the activity of lymphocytes, thereby strengthening human immunity;
• is an antioxidant;
• promotes the removal of dead cellular structures and cell waste products from the body;
• prevents the development of a number of ophthalmological diseases, is a structural element of the vitreous body of the human eye and is part of other elements of the visual apparatus, promotes the passage of light rays to the retina, while preventing their distortion;
• prevents the occurrence of joint disorders;
• is a modulator of facial and body contours;
• has the ability to retain moisture in the skin, gives the skin elasticity, increases its resistance to the influence of adverse factors and prevents the appearance of age-related and facial wrinkles;
• has a beneficial effect on the functioning of the reproductive system;
• participates in the processes of intrauterine development and fetal growth during pregnancy.

It is worth noting that this compound also plays a significant role in the process of egg fertilization. Normally, an oocyte released from the ovary during the ovulation period is covered with two protective membranes (zona pellucida and corona radiata) containing a large amount of hyaluronate. Fertilization is possible only if the integrity of these membranes is not broken. When the protective layers are destroyed, the egg loses its ability to be fertilized by sperm and dies. In other words, insufficient intake of hyaluronate into the body can cause female infertility.

What products contain hyaluronic acid?

In youth, the human body is able to synthesize hyaluronic acid and independently satisfy its need for this substance. However, with age, the production of this compound decreases, and its deficiency begins to have a negative effect on the condition of the skin, joints, and the functioning of internal organs and systems. One of the ways to eliminate the unpleasant symptoms that accompany a lack of hyaluronate is to include in the menu foods rich in this substance or compounds that stimulate its production.

Meat products are considered the main food source of hyaluronic acid. Moreover, the greatest amount of this substance is present in those types of meat (and dishes prepared on their basis) that contain a sufficient amount of joints, tendons, cartilage and skin. For example, you can replenish lost reserves of hyaluronate by regularly including in the menu:

• rich meat broths;
• boiled or stewed meat on the bone;
• jellied meat made from turkey, pork, chicken or beef;
• any dishes containing gelatin (jelly, marmalade, marshmallows, etc.).

It is worth noting that plant foods are also a rich source of hyaluronic acid. In particular, increased concentrations of this substance were found in soybeans, soy milk and vegetables containing large amounts of starch. At the end of the 20th century, substances that stimulate the production of hyaluronate were discovered in the skin of red grapes. As a result, red wines and natural grape juice were included among the plant products that help replenish the reserves of this unique compound in the human body.

Significant amounts of hyaluronic acid are also found in some medicinal herbs. In particular, the leaves and fruits of burdock, which are used to prepare healthy and tasty herbal teas, are recognized as a rich source of this substance.

What factors influence the synthesis and absorption of hyaluronate in the body?

There are several factors that can have both positive and negative effects on the processes of production and absorption of hyaluronic acid. For example, the synthesis of this compound and its digestibility are significantly increased with the simultaneous consumption of foods enriched with ascorbic acid and rutin. For this reason, nutritionists recommend that people suffering from a lack of hyaluronic acid include the following foods and dishes in their diet as often as possible:

• green tea;
• citrus fruits (grapefruits, oranges and lemons are best);
• rowan;
• some berries (blackberries, blackcurrants, raspberries);
• walnuts;
• apricots;
• cherries;
• greens (parsley, cilantro, dill);
• all varieties of cabbage;
• leaf salad;
• rose hips and infusions prepared on its basis;
• tomatoes.

At the same time, there are factors that can significantly slow down the processes of production and absorption of hyaluronic acid. They are the main reasons for the development of deficiency of this substance in the body.

Lack of hyaluronic acid and its consequences

The main reasons for the formation of hyaluronic acid deficiency in the body are:

• smoking;
• abuse of high-strength alcoholic beverages, consumption of red wine in doses exceeding the permissible limits (more than 140 ml during the day);
• insufficient intake of vitamin C, rutin and other nutrients;
• excessively long stay in the solarium, under the influence of direct sunlight, refusal to use sunscreen;
• age-related decrease in the concentration of this substance in the tissues of the human body.

A deficiency of this compound can lead to a wide range of adverse effects. In particular, signs of hyaluronate deficiency may include:

• deterioration in general well-being, fatigue, indifference to current events;
• weakening of the body’s immune forces, frequent occurrence of colds;
• dehydration, sagging, excessive dryness of the skin;
• change in the contours of the face and body for the worse;
• development of dermatological diseases;
• deterioration of vision and the appearance of other disturbances in the functioning of the visual apparatus;
• early appearance of wrinkles and other signs of aging;
• the development of joint diseases and the occurrence of other pathologies in the musculoskeletal system;
• prolonged healing of wounds, slow fusion of bone tissue in fractures;
• the appearance of signs of intoxication of the body;
• inability to conceive a child for a long time;
• the appearance of disturbances in the intrauterine development of the fetus, slowing down its growth.

If such symptoms are detected, it is necessary to reconsider your diet and enrich it with foods rich in hyaluronic acid and substances that activate its synthesis. In addition, it is necessary to give up bad habits and protect yourself as much as possible from the effects of factors that negatively affect the production of this essential compound.

The first mention of an unusual polysaccharide with a high molecular weight, which was isolated from the vitreous humor of the bovine eye, was made in 1934 by German biochemists Karl Meyer and John Palmer. It was they who proposed calling the new substance hyaluronic acid. But back in 1918, Levene and Lopez-Suarez isolated a polysaccharide consisting of glucosamine, glucuronic acid and a small amount of sulfate ions from the vitreous body and umbilical cord blood. Then its name was mucoitin - sulfuric acid, but it has now been established that it was hyaluronic acid, isolated with an admixture of sulfated glycosaminoglycans.

Over the next 10 years, K. Meyer and a number of other scientists isolated hyaluronic acid from animal organs. In 1937, F. Kendall isolated hyaluronic acid from streptococcal capsules.

The first experience of using HA in medicine dates back to 1943, when the Soviet doctor Nikolai Fedorovich Gamaleya used it in complex dressings for frostbitten Red Army soldiers in a military hospital. The extract from the umbilical cord, which he called the “regeneration factor,” was approved by the USSR Ministry of Health as the “Regenerator” drug. Also, the Hungarian scientist Andre Balazs, since 1947, studied the viscosity of HA depending on the pH and ionic strength of the solution, its breakdown under the influence of ultraviolet radiation, and also studied how hyaluronic acid acts on living cells.

Currently, hyaluronan as an object of research can be found in biochemistry, molecular biophysics, bioorganic and radiation chemistry. Medical aspects include studying the role of hyaluronic acid in fertilization, embryogenesis, the development of an immune response, wound healing, cancer and infectious diseases, the aging process and in solving problems of aesthetic medicine. A wide range of practical applications of hyaluronic acid promotes epithelial regeneration, prevents the formation of granulation tissues, adhesions, scars, reduces swelling, reduces skin itching, normalizes blood circulation, promotes scarring of trophic ulcers, and protects the internal tissues of the eye. Hyaluronic acid is used quite well in applied biochemistry and enzymology as a substrate for the quantitative determination of hyaluronidase enzymes.

What exactly is hyaluronic acid? It is a long, unbranched molecule in which D-glucuronic acid and N-acetylglucosamine residues alternate. Without going into details, we note that both of these substances are modified glucose molecules. A hyaluronic acid molecule can contain more than 30,000 residues of each of these substances. In addition, in the body this chain is always associated with some amount of protein. Interestingly, such a structure is universal and is found in a wide variety of representatives of the animal world and even in some bacteria. Hyaluronic acid belongs to the class of glycosaminoglycans.

Figure 1. Structure of hyaluronic acid

Previously, methods were used to obtain hyaluronic acid from the vitreous body of a cow's eye and the comb of a rooster. The disadvantages of these production methods were their high cost and the presence of protein impurities in the final product, which led to a large number of allergic reactions to the drug.

Modern HA production is based on a fermentation process using bacteria (Streptococcus equi and Streptococcus zooepidemicus). HA obtained in this way has a higher degree of purification, which explains the better tolerability of HA by patients. The biotechnology of producing hyaluronan from bacterial strains of producers involves cultivating them under selected conditions, under which, at the stage of logarithmic growth, a capsule of polysaccharide is formed on the surface of bacterial cells, and at the stationary stage of growth, HA can be secreted into the culture liquid, the capsule becomes thinner or completely disappears.

HA is sensitive to acid-base hydrolysis. Even slight acidification of a HA solution with acetic acid leads to an irreversible decrease in viscosity by 2.5 times. HA is completely hydrolyzed by mineral acids to glucuronic acid, glucosamine, acetic acid and carbon dioxide. Dilute sulfuric acid hydrolyzes the acid in a short time to form disaccharide crystals.

Redox depolymerization of hyaluronan . The destruction of the polysaccharide macromolecule under the influence of redox environments occurs through a free radical mechanism. Free radicals are formed with the participation of ascorbic acid, hyaluronan and oxygen. It has been proven that hyaluronic acid is depolymerized by the action of iron ions in the presence of ascorbic acid. Consequently, HA isolated in an atmosphere of nitrogen or argon has a higher degree of polymerization compared to that isolated in air.

For medical use, sterilization of hyaluronan solutions is necessary. It is carried out by autoclaving at a temperature of 120-130ºС or by ionizing gamma radiation. In both cases, significant depolymerization of the biopolymer and loss of its original therapeutic activity occurs. There are known methods for protecting hyaluronan solutions from depolymerization, based on adding various amino acids, boric acid and glycerin, hydroquinoline sulfate, uric acid, and phenolic compounds (pyrogallol) to the solutions.

The characteristic properties of hyaluronic acid - its pronounced biological activity, excellent biocompatibility, lack of antigenicity, irritation and other side effects - have attracted the attention of scientists. Thanks to its unique physicochemical properties, HA has found application in various fields of medicine, cosmetology and veterinary medicine. The fact that HA is part of many tissues (skin, cartilage, vitreous body) and is organ-specific and species-nonspecific determines its use in the treatment of diseases associated with these tissues.

The biological functions of hyaluronic acid can be divided into “passive” and “active”. As an inert material, HA is involved in tissue homeostasis, in the steric regulation (osmosis) of the penetration of any substances, acts as a “lubricant” that improves joint mobility, etc. The “active” functions of HA consist in specific binding to proteins in the extracellular matrix and on the cell surface. This interaction plays an important role in the formation of cartilage tissue, in the processes of cell proliferation, in the morphogenesis and embryonic development of animals, as well as in the mechanisms of inflammation and cancer.

Hyaluronic acid is used in oncology as a therapeutic agent. The mechanisms of action of GC on tumor cells are varied. At the molecular level, the mechanism is that high molecular weight HA, by binding to receptors on the cell membrane of tumor cells, slows down their migration and the formation of metastases. The second mechanism of action is that the administration of high molecular weight HA promotes the formation of a connective tissue capsule around the tumor. The third mechanism is associated with the property of the high molecular weight fraction to inhibit tumor vascularization (the growth of blood vessels into the tumor) and thereby lead to a slowdown in tumor growth and metastasis, while the low molecular weight fraction, on the contrary, induces.

Hyaluronic acid has proven itself quite well in the healing of burn wounds, ulcers, scars and postoperative interventions. Scientists have found that it does not have an irritating effect, but on the contrary, causes an anti-inflammatory effect and promotes rapid tissue regeneration. In an experiment, a bioexplant (film) based on oxidized HA showed accelerated healing of high-risk intestinal anastomotic sutures.

HAs are used in the preparation of pharmaceutical compositions as thickeners, lubricants, agents for film coatings resistant to gastric juice, in particular in the preparation of capsules, gels, colloids and various devices (for example, contact lenses, gauze items, etc.) . Probably, the mechanism of accumulation of a number of drugs and antibiotics in connective tissue structures is based on their binding to tissue proteoglycans. The same can be said about the mechanisms of deposition in tissues, especially in the connective tissue matrix, of various pathological products. Normally, on the first day of wound healing, there is an increase in the concentration of HA, which binds to the fibrin network and forms a transition matrix that stimulates the activation and migration of granulocytes, macrophages and fibroblasts, and the proliferation of epithelial cells. In addition, HA, by enhancing phagocytosis, promotes more complete cleansing of the wound from necrotic elements. Due to increased activity of macrophages, the formation of trophic factor increases, which attracts fibroblasts and endothelial cells to the affected area.

The content of hyaluronan in human skin is not constant. There are minor seasonal fluctuations in HA in the dermis: in summer the level of hyaluronan is slightly lower than in winter. This is associated with an increased rate of degradation of HA under the influence of UV radiation. The most significant age-related decrease in GC concentration is observed. Starting from the age of 60, there is a multiple decrease in the concentration of HA in the dermis. Therefore, intracellular injection of native HA seems to be a completely natural way of inflaming its deficiency. This injection method in aesthetic medicine is called biorevitalization.

In the scientific literature you can find extensive information about the chemical structure, macromolecular characteristics, biological properties and medical uses of hyaluronic acid.

HA is part of the main intercellular substance of connective, epithelial and nervous tissues; it is present in large quantities in the vitreous body of the eye, synovial fluid of joints, skin, walls of arteries and veins, heart valves, and in the glomerular basement membrane of the kidneys.

Since the discovery of hyaluronic acid, there has been a significant evolution of views. If at first it was believed that this polysaccharide serves as a passive structural component of the intercellular matrix, then by now it is involved in many biological processes: from reproduction, migration, differentiation of cells during embryogenesis to the regulation of inflammation and wound healing, metastasis of cancer cells. In the body, HA performs multiple physiological functions: it serves as the basis for the functioning of the body system, determines the permeability of tissues and vessels of the circulatory system, and resistance to infection. But with age, all functions slow down.

Such a wide variety of biological properties of hyaluronic acid is due to the function of molecular weight, which plays a significant role in cell behavior, polymorphism of structural forms and physicochemical properties of molecules of different molecular weights, depending on the ionic environment and concentration of the biopolymer in tissues and organs.

To summarize, we can say that hyaluronic acid has found its use in many branches of medicine. It is used in cosmetic injections (biorevitalization) and is included in various cosmetics. It should be noted that HA can also have negative consequences when frequently injected under the skin. To maintain your skin tone, you need to lead a healthy lifestyle, eat right and not abuse bad habits. Ophthalmologists also use it as a treatment for cataracts and dry eye syndrome. In immunology, it is used for the complex treatment of immunodeficiency states due to viral infections. It can also be used to treat gastric and duodenal ulcers by activating trypsin.

Bibliography

1. Egorov E.A. Hyaluronic acid: application in ophthalmology and treatment of dry eye syndrome // Breast Cancer. Clinical ophthalmology. – 2013. – Volume 13, No. 2. S. – 72.
2. Sigaeva N.N., Kolesov S.V., Nazarov P.V., Vildanova R.R. Chemical modification of hyaluronic acid and its use in medicine // Bulletin of the Bashkir University. – 2012. – T.17. No. 3. S. – 1221 – 1222.
3. Strelnikova L.N., Kleshchenko E.V., Astrin A.V. Chemistry and life // Monthly scientific and population magazine. – 1.12.2010. No. 12. S. – 22 – 23.
4. Khabarov V.N., Boykov P.Ya., Selyanin M.A. Hyaluronic acid: production, properties, application in biology and medicine. – M.: Practical Medicine, 2012. – 224 p.: ill. S. – 9 – 11, 19 – 30, 218.

Hyaluronic acid (hyaloid = glassy + uronic = acid) is a substance belonging to the group of polysaccharides, synthesized by the cells of most living organisms, and is an important component of the skin, muscles, nerves and other human tissues.

In descriptions of the compositions of cosmetic products, it is sometimes called “hyaluron”; biochemists more often use the phrase “sodium hyaluronate”, since in the human body it is present mainly in the form of sodium salt.

Biological role

Hyaluronic acid is necessary for the formation of the intercellular substance, which is the medium for the functioning of cells: their division, the supply of nutrients to them, and the removal of waste products.

Half of all hyaluronic acid in the body is found in the skin. Here it is a natural filler of the gaps between the fibrous elements of the skin - collagen and elastin, and participates in their synthesis.

Hyaluronic acid is also involved in wound healing processes, affects immune reactions, blocks the effect of free radicals on cells, protecting tissues from premature aging.

One of the most important properties of hyaluronic acid is its highest hydrophilicity - the ability to bind moisture. One molecule of it can hold up to 500 molecules of water. Even a 1% aqueous solution of hyaluronic acid is no longer a liquid, but a viscous gel.

What does hyaluronic acid deficiency lead to?

With age, under the influence of environmental factors and natural aging processes, the content of hyaluronic acid in the human body decreases; by the age of 50, its amount is halved. Decrease in concentration of g.c. in the skin leads to dehydration, a decrease in the synthesis of collagen and elastin in it, which manifests itself in the form of dryness, sagging, and the appearance of wrinkles.

Application in cosmetology

In modern cosmetology, hyaluronic acid is used as the main component of preparations to moisturize the skin. A more effective substance for this purpose has not yet been found.

Since hyaluronic acid is not a foreign substance to the body, preparations based on it are hypoallergenic.

Hyaluronic acid used in cosmetology can be of either natural or artificial origin. Since its metabolism is very active (the gk molecule “lives” in the body for 2-3 days, then it is destroyed and a new one is synthesized by the cells), artificially synthesized substances, distinguished by the fact that they contain g molecules, are more often used for introduction into the deep layers of the skin. To. “stitched” together and the body requires more time to break them down.

In products for external use (creams, emulsions, lotions, etc.), hyaluronic acid acts as a moisturizer. The thinnest film formed on the surface of the skin prevents excessive evaporation of water and retains the necessary moisture. At the same time, it does not clog skin pores, does not disrupt transcutaneous gas exchange, and promotes deeper penetration of other active substances included in the product. But, applied to the surface of the skin, hyaluronic acid does not penetrate into its deeper layers and provides only superficial, short-term hydration.

For deep and long-term hydration, to stimulate fibroblasts, the introduction of hyaluronic acid into the deep layers of the skin is used - method biorevitalization .

Preparations with a high concentration of hyaluronic acid, in gel form, are used in contour plastic surgery– for correcting nasolabial folds, wrinkles, increasing lip volume.

Mesotherapy preparations use the property of hyaluronic acid to improve the penetration into cells of other substances introduced along with it.

Hyaluronic acid is used not only in cosmetology, it is part of drugs widely used in many areas of medicine - ophthalmology, cardiology, transplantology, surgery, etc.

In the late 80s of the twentieth century, doctors noticed that the process of wound healing in the prenatal period occurs somewhat differently than after birth. To treat congenital malformations, surgical operations were performed on fetuses located inside the body of pregnant women (at 2-6 months of pregnancy). After birth, no traces of the operations performed were found on the bodies of these children. Scientists explain this by the very high concentration of hyaluronic acid in the fetal body and the amniotic fluid surrounding it.

Hyaluronic acid! There is a lot of talk about it; it is included in the formulations of new skin care products. All cosmetics manufacturers claim to use the best types of hyaluronic acid in their products. But what is hyaluronic acid, what does it do, how does it work, and what type is considered the best?

Hyaluronic acid (HA) is the most important factor in skin hydration. This molecule forms a three-dimensional network that acts like a sponge and literally traps water around and inside its folds.

In addition, HA is used by the body as a lubricant in joints, the auricle is mainly composed of it, and it is also one of the structural polymers of the vitreous body of the eye. HA can stimulate or inhibit inflammation, promote wound healing and skin restoration. It is an important component of the intercellular substance of literally all connective tissue of the human body.

In the skin, HA is found primarily in the basement membrane of the epidermis and dermis, maintaining the space between cells, moisturizing and facilitating the passage of nutrients.

The body of a woman weighing 60 kg contains about 13 g of hyaluronic acid, 4.3 g of this amount is processed and renewed every day.

However, before discussing how HA works and what it can do for the skin, it would be a good idea to first present a short dossier on this substance to better understand its mode of action.

Hyaluronic acid 101

Hyaluronan, or hyaluronic acid, is a natural polymer, that is, a large molecule made up of many repeating small molecules called “subunits.”

In the case of hyaluronic acid, this subunit is the disaccharide D-glucuronic acid and N-acetyl-D-glucosamine bound together.

The length of a HA molecule can range from 2 to 25 thousand disaccharides. The molecular weight of this natural polymer ranges from 800 to 2,000,000 Daltons (Da), with an average molecular weight of HA of 3 MDa in joints and about 2 MDa in skin.

The body continually synthesizes and breaks down HA (as mentioned above, the body completely replaces HA approximately every three days). As large HA molecules gradually degrade, fragments of very different molecular weights are formed. A set of these fragments - from 800 Da to 2 MDa - is present at any time in normal tissues.

Based on the size of the HA molecules, they are divided into different fractions.

• Very high molecular weight: 3–20 MDa.
• High molecular weight: ~ 2 MDa.
• Average molecular weight: ~ 1 MDa.
• Low molecular weight: ~300 kDa.
• Very low molecular weight: ~60 kDa.
• Oligomers: from 800 Da to 10 kDa.

Appearance and biological effects

It is clear that molecules, whose molecular mass can vary up to 12,500 times, look and behave completely differently in biological systems, producing different biological effects. This has been shown in more detail in numerous studies conducted in recent years.

It is commonly said that HA can absorb 1000 times its own weight in water. However, this only applies to high molecular weight HA, and those with a lower molecular weight are obviously able to absorb much less water.

Therefore, in practice, if you take 1% high molecular weight HA in water, you can get a rather viscous liquid or liquid gel. Low molecular weight HA at the same concentration will be a much less viscous liquid or a completely watery gel, while the oligomer will be as liquid as water. Needless to say, HA with a molecular weight of 20 MDa will be a very thick gel in this case.

You may wonder why there is so much information about the size of the molecules and the appearance of the gel. The answer is quite interesting. HA with a molecular weight of 3–20 MDa, that is, high molecular weight HA, is the type of HA found in cellulite. This is an abnormally large size of HA molecules, due to which water is firmly retained in the subcutaneous fatty tissue, which in turn contributes to the manifestation of visible signs of cellulite.

Therefore, the presence of HA with too high a molecular weight in tissues is undesirable - this is a sign of a pathological process. On the other hand, the presence of too many HA fragments in tissues, that is, too many oligomers or even 20 kDa HA, is also undesirable, since they are known to stimulate inflammation. However, even inflammation has a right to exist in some situations, and sometimes it is necessary (for example, when healing wounds).

All other molecular weights (50 kDa - 2 MDa) appear to be neutral or beneficial, with 2 MDa considered the most "normal" (so to speak) and inhibits inflammation.

So we can say that the only truly “bad” type of HA is the extremely high molecular weight HA, which also promotes fibrosis.

Diet, lifestyle and hyaluronic acid

A diet rich in vegetables (magnesium) and fruits (ascorbic acid) is thought to help increase the body's natural synthesis of GC. Also, some foods are rich in HA or its precursors. Examples include bone broth, organ meats, and joint cartilage.

Hyaluronan can also be taken orally as a dietary supplement and actually reaches the skin and joints to help increase hydration, keep them looking youthful longer, and maintain health. This is similar to taking hydrolyzed collagen orally, which also helps delay skin aging and maintain the firmness and elasticity of ligaments and tendons.

Ultraviolet radiation reduces the HA content of the skin, which leads to dryness and inflammation. By providing the skin with a sufficient amount of HA in the summer, including from the inside, we can be sure that it will be able to remain hydrated and protected from sun rays.

There is no specific food that has a proven ability to increase the body's own HA synthesis, but drinking a certain amount of drinking water daily will promote hydration, since the water molecule is no less important for this than hyaluronic acid, because without water, hyaluronan is absolutely useless. Ideally, you should drink two liters of water per day. Thus, to improve skin hydration and obtain a rejuvenating effect, it is advisable to combine the oral use of HA in the form of dietary supplements and drinking a sufficient amount of water. For maximum results, you can additionally use a high-quality serum, gel or cream containing HA.

Dermal absorption of HA from cosmetic formulations

Since HA stimulates skin repair and hydration, and the skin produces less and less of it as we age, it goes without saying that you want to add some HA to your skin in the form of a beauty serum, cream or gel.

It is clear that HA with a large molecular weight will not even be able to penetrate the epidermis, while everything below 300 kDa penetrates the dermis and even subcutaneous fat. The lower the molecular weight, the deeper HA can penetrate the skin.

However, not all so simple. As we mentioned above, you need to understand why we use lower molecular weight HA in our formulation. By “pushing” HA with a molecular weight of 20 kDa into the skin, we do not solve all skin problems, since this can be both beneficial and irritating for the skin. When using HA with an extremely low molecular weight, things become even more complicated.

However, most research studies have shown that HA molecules with a molecular weight somewhere between 50 and 300 kDa penetrate well into the skin and have beneficial effects on it. My personal experience also suggests that this is the best molecular weight range to use.

HA with a molecular weight of 1 MDa can hydrate the epidermis itself without penetrating further, while a molecule with a molecular weight of 2 MDa simply sits on the surface of the epidermis and does not go anywhere else. On the other hand, I have found that the 10 kDa HA is not as beneficial and can be irritating to the skin in high concentrations, as evidenced in the scientific literature.

This different absorption capacity of HA, depending on its molecular weight, is the reason that more and more cosmetic companies are now using HA of different molecular weights in their formulations.

HA molecules can also be linear or cross-linked. The linear molecule is the standard HA found in humans and in nature. Cross-linked HA is a human invention and is a more stable form of HA with a higher hydrating capacity. But unfortunately, cross-linked HA has less ability to penetrate the skin because the molecule is “thicker” and cannot easily cross the epidermis.

Cross-linked HA is used as a filler in mesotherapy, but today it can also be found in some anti-aging creams.

Serums, gels and creams

It is quite easy to make a cosmetic serum or gel using HA and water. However, creams are a different matter. Here, HA can greatly increase the instability of the emulsion system. Therefore, most HA products on the market are gels and serums, which are easier to obtain.

Many HA cosmetics on the market contain about 0.1% of this substance, that is, 1 part HA and 999 parts water and some other ingredients. However, more concentrated products may contain up to 2% HA. Higher concentrations are not practical because the cream or gel becomes too thick and uncomfortable.

Hyaluronic acid is currently one of the most popular and important ingredients for anti-aging skin and facial care. It can also be found in some body care products. Unfortunately, unless the type and concentration of HA is specifically mentioned on the packaging of a cosmetic product, it is very difficult to understand what exactly is used and in what concentrations, but this applies to all ingredients in cosmetic formulations.

On the other hand, some skin care products include active ingredients that increase the skin's own HA synthesis. This gives a slightly delayed result, but bypasses the problem of HA absorption, since the “own” HA is synthesized within the skin. Other active substances used in cosmetics can inhibit the action of HA-degrading enzymes, hyaluronidases, in the human body (most polyphenols have this activity). This prolongs the useful life of HA in the skin and suppresses its early or excessive degradation.

What is the origin of HA for cosmetics?

Once upon a time, HA of animal origin, obtained from pig ears or rooster combs, was used in cosmetics. I remember that the first HA we purchased for use in our products in 2002 was derived from piglets.

Today, HA is produced by bacterial fermentation, which produces a standard molecular size of 2 MDa. It is then “cut” either by enzymes or by hydrolysis to produce smaller molecules. The same thing happens in the human body - HA with a molecular weight of 2 MDa is cut into smaller pieces by enzymes called hyaluronidases.

Hyaluronidase and cellulite

Sometimes, to destroy the HA with an excessively high molecular weight, which we mentioned above, doctors inject hyaluronidase into the tissue.

One such use of hyaluronidase is to temporarily reduce the appearance of cellulite. I use the word “temporary” because the human body can restore the molecular weight of newly synthesized HA to 20 MDa in just a few days.

Therefore, a long-term solution to cellulite cannot be achieved with hyaluronidase injections. These should be measures primarily aimed at reducing water retention in tissues and reducing the synthesis of HA with a molecular weight of 20 MDa. But that's a story for another article...

Conclusion

As we age, the human body synthesizes less and less HA, which is why it is necessary to protect the existing HA and increase its content in the skin.

This can be done by avoiding excessive sun exposure; with a diet rich in vegetables, herbs, and organ meats; drinking enough water; using good HA-based skin care cosmetics with molecules of different molecular weights, ideally from 50 to 300 kDa.

Dietary supplements with hyaluronic acid also help because they actually have a beneficial effect on the skin (and joints), helping to hydrate and nourish the body from the inside out.

Hyaluronic acid [HA] is found in the extracellular matrix of vertebral tissues, in the surface coating of certain Streptococcus species and Pasteurella bacterial pathogens, and on the surface of some partially virus-infected seaweeds. Hyaluronic acid synthases [HAS] are enzymes that polymerize HA using UDP sugar precursors that are found in the outer membranes of these organisms. GCS genes from all the above-mentioned sources were identified. There appear to be two distinct classes of GCS based on differences in amino acid sequence, predicted membrane topology, and proposed reaction mechanism.

All GCS were identified as class I synthases, with the exception of GCS in Pasteurella species. The catalytic mode of operation of the only class II GCS (pmGCS) was also explained. This enzyme extends outer HA-attached oligosaccharide acceptors by adding individual monosaccharide units to the non-reducing end to form long polymers in vitro; no Class I GCS has this capability. The mode and direction of HA polymerization catalyzed by class I GCS remain unclear. The PMGC enzyme was also analyzed for its two activities: GlcUA transferase and GlcNAc transferase. Thus, two active sites exist in one PMGC polypeptide, refuting the widely accepted dogma of glycobiology: “one enzyme, one modified sugar.” Preliminary evidence suggests that class I enzymes may also have two sites of activity.

The catalytic potential of the PMGC enzyme can be used to create new polysaccharides or engineer oligosaccharides. With so many potential GC-based medical treatments available, this chemoenzymatic technology promises to benefit our quest for good health.

Keywords

Hyaluronic acid (HA), chondroitin, glycosyltransferase, synthase, catalysis, mechanism, chimeric polysaccharides, monodisperse oligosaccharides

Introduction

Hyaluronan [HA] is a very rich glycosaminoglycan in vertebrates, having both structural and signaling roles. Certain pathogenic bacteria, namely groups A and C of Streptococcus species and type A of Pasteurella multocida, produce an extracellular covering of HA called a capsule. In both species, the HA capsule is the toxicity factor that provides the bacteria with resistance to phagocytes and complementarity. Another organism that produces HA is the seaweed Chlorella, which is infected with a certain large double-stranded DNA virus, PBCV-1. The role of HA in the life cycle of this virus is not yet clear at this time.

Illustration 1. HA biosynthesis reaction.

Enzymes of the class of glycosyltransferases that polymerize HA are called GC synthases (or GCS), according to the old terminology that also includes GC synthetases. All known HA synthases are variants of a single polypeptide responsible for the polymerization of the HA chain. The UDP sugar precursors, UDP-GlcNAc and UDP-GlcUA are used by GC synthases in the presence of a divalent cation (Mn and/or Mg) at neutral pH (Fig. 1). All synthases are membrane-bound proteins in the living cell and are found in the membrane fraction after cell lysis.

Between 1993 and 1998, GK synthases of groups A and C of Streptococcus [spGCS and seGCS, respectively], vertebrate GK synthases [GCS 1,2,3], algal virus GK synthase [svGCS], and also GK synthase type A of the species Pasteurella multocida [pmGKS]. The first three types of GK synthases appear to be very similar in size, amino acid sequence, and predicted membrane topology. In contrast, the GK synthase from Pasteurella species is larger and has a significantly different sequence and predicted topology from other synthases. Therefore, we hypothesized the existence of two classes of HA synthases (Table 1). Class I enzymes include streptococcal, vertebrate, and viral proteins, while the Pasteurella species protein is currently the only member of class II. We also have some evidence that the catalytic processes of class I and class II enzymes are different.

Table 1. Two classes of GK synthases:

Although Pasteurella spp. GK synthase was the last enzyme to be discovered, several features of pmGXS have contributed to significant advances in its study compared with some members of the class I enzymes, which have been studied for four decades. The key feature of pmGCS, which made it possible to clarify the molecular direction of polymerization and the identification of its two active sites, is the ability of pmGCS to extend the externally located acceptor oligosaccharide. Recombinant pmGCS adds single monosaccharides in a repetitive manner to a GC-associated oligosaccharide in vitro. An intrinsic feature of each monosaccharide transfer is responsible for forming an alternative repeat of disaccharides in that glycosaminoglycan; simultaneous formation of the disaccharide unit is not required. On the other hand, no such extension of external acceptors has been demonstrated for any class I enzyme. Through basic science research, we have now developed some biotechnological applications of a remarkable class of GK synthase protein from the Pasteurella species.

Materials & Methods

Reagents

All reagents for molecular biology studies without special marking were from Promega. Standard oligonucleotides were from Great American Gene Company. All other high purity reagents, unless otherwise noted, were from Sigma or Fisher.

Truncation of pmGCS and point mutants

A series of truncated polypeptides were produced by amplification of the pPm7A insert by polymerase chain reaction with Taq polymerase (Fisher) and synthetic oligonucleotide primers corresponding to different parts of the PMGCs, with an open reading frame. The amplicons were then cloned into the expression plasmid pKK223-3 (tac promoter, Pharmacia). The resulting recombinant constructs were transformed into Escherichia coli strain TOP 10F" cells (Invitrogen) and grown on LB medium (Luria-Bertani) with ampicillin selection. Mutations were made using the QuickChange site-directed mutagenesis method (Stratagene) with the pKK/pmGCS plasmid as DNA sample.

Enzyme preparation

To prepare the membrane containing recombinant full-length pmGKC, pmGK1-972 was isolated from E. coli as described. For the soluble truncated pmGKS proteins, pmGKS1-703, pmGKS1-650 and pmGKS1-703 - containing mutants, cells were extracted using B-PerTM II Bacterial Protein Extraction Reagent (Pieree) according to the manufacturer's instructions, except that the procedure was performed at 7°C in the presence of protease inhibitors.

Enzymatic pathways for HA polymerization. GlcNAc modification or GlcUA modification

Three variants were designed to detect whether (a) polymerization of long HA chains occurs, or (b) addition of a single GlcNAc to a GlcUA-terminal HA acceptor oligosaccharide, or (c) addition of a single GlcUA to a GlcNAc-terminal HA acceptor oligosaccharide. The total activity of GCS was assessed for a solution containing 50 mM Tris, pH 7.2, 20 mM MnCl2, 0.1 M (NH4)2SO4, 1 M ethylene glycol, 0.12 mM UDP-(14C)GlcUA (0.01 μCi; NEN), 0.3 mM UDP- GlcNAc and a different set of HA oligosaccharides obtained from testicles by treatment with hyaluronidase [(GlcNAc-GlcUA)n, n= 4-10] at 30°C for 25 minutes in a reaction volume of 50 μl. GlcNAc-transferase activity was assessed for 4 minutes in the same buffer system with a different set of GC oligosaccharides, but with only one sugar as a precursor - 0.3 mM UDP-(3H)GlcUA (0.2 μCi; NEN). GlcUA transferase activity was assessed for 4 minutes in the same buffer system, but with only 0.12 mM UDP-(14C)GlcUA (0.02 μCi) and with an odd set of GC oligosaccharides (3.5 μg uronic acid) prepared by exposure to acetate mercury on GK-lyase of Streptomyces. Reactions were terminated by adding SDS to 2% (w/v). Reaction products were separated from substrates by paper (Whatman 3M) ​​chromatography with ethanol/1 M ammonium sulfate, pH 5 5, as the main solvent (65:35 for GCS and GlcUA-Tase assessment; 75:25 for GlcNAc-Tase assessment). To evaluate HA, a paper strip sample was rinsed with water, and the association of radioactive sugars into the HA polymer was detected by liquid scintillation calculated using the BioSafe II cocktail (RPI). For half-test reactions, the sample and downstream 6 cm stripes were counted in 2 cm increments. All evaluation experiments were scaled to be linear with respect to incubation time and protein concentration.

Gel filtration chromatography

The size of the HA polymers was analyzed chromatographically on Phenomenex PolySep-GFC-P 3000 columns, elution was carried out with 0.2 M sodium nitrate. The column was standardized with fluorescent dextrans of different sizes. Radioactive components were detected using an LB508 Radioflow sensor (EG&G Berthold) and a Zinsser cocktail. Compared to the full GC evaluation using paper chromatography described above, these 3-minute reactions contained twice the UDP-sugar concentrations, 0.06 μCi UDP-(14C)GlcUA, and 0.25 nanograms of the GC series of oligosaccharides. Additionally, the addition of boiling (2 minutes) ethylene diamine tetracylic acid (final concentration 22 mM) was used to complete the reactions instead of adding SDS.

Results and discussion

Utilization and specificity of the GCS acceptor

Several oligosaccharides have been tested as acceptors for recombinant PMGKS1-972 (Table 2). HA oligosaccharides were obtained from testicles by hyaluronidase cleavage and elongated by pmGCS using suitable UDP sugars delivered. Reduction with sodium borohydrate does not interfere with the acceptor activity. On the other hand, oligosaccharides obtained from HA by lyase cleavage do not support elongation; the dehydrated unsaturated unreduced terminal residues of GlcUA require hydroxyl groups that can attach the incoming sugar from the UDP precursor. Therefore, pmGX-catalyzed extension occurs in the case of unreduced end groups. In a number of parallel experiments, recombinant forms of class I synthases, spGCS and x1GCS, were discovered, which do not extend the GC-derived acceptors. Considering the direction of activity of class I enzymes, conflicting reports have been made and further research is needed.

Table 2. Specificity of oligosaccharide acceptors PMGCS:

Interestingly, chondroitin sulfate pentamer is a good acceptor for PMGCs. Other structurally related oligosaccharides, such as chitoterose or heparosan pentamer, however, do not serve as acceptors for pmGCS. Overall, pmGCS appears to require β-linked GlcUA-containing acceptor oligosaccharides. We hypothesize that the oligosaccharide binding site is intermediate in the HA retention chain during polymerization.

Molecular analysis of PMGCS transferase activity: two active sites in one polypeptide

The ability to measure two components of the glycosyltransferase activity of GK synthase, GlcNAc transferase and GlcUA transferase, was made possible by molecular analysis of pmGCS. We noted that a short duplicated sequence motif: Asp-Gly-Ser (Aspartic k-ta-Glycine-Serine) was present in pmGCS. From a comparison analysis of the hydrophobic groups of many other glycosyltransferases that produce β-linked polysaccharides or oligosaccharides, it has been suggested that, in general, there are two types of domains: the “A” and “B” regions. PmGKS, a class II synthase, is unique in that it contains two “A” domains (personal communication, B. Henrissat). It has been proposed that certain members of class I GK synthases (spGCS) contain single "A" and single "B" regions. Various deletion or point mutants of pmGCS were assessed for their ability to polymerize GC chains or their ability to add a single sugar to a GC acceptor oligosaccharide (Table 3). To summarize, pmGCS contains two different active sites. Mutagenesis of the DGS aspartate motif (residue 196 or 477) at both sites resulted in loss of HA polymerization, but the activity of the other site remained relatively unaffected. Thus, the dual activity of GC synthase was converted into two different single glycosyltransferase activities.

Table 3. Activity of pmGCS with a deleted region or point mutation.

Removal of the last 269 residues from the terminal carboxyl group converted a weakly expressed membrane protein into a highly expressed soluble one. Examination of the amino acid sequence of the PMGC protein in this region, however, does not reveal typical secondary structure features that would provide direct interaction of the enzyme with the lipid bilayer. We hypothesize that the terminal carboxyl group of the catalytic enzyme pmGCS docks with the membrane-bound polysaccharide transport apparatus of the living bacterial cell.

The first "A" region of the pmGCS, A1, is a GlcNAc pelvis, while the second "A" region, A2, is a GlcUA pelvis (Fig. 2). This is the first identification of two active sites for an enzyme that produces a heteropolysaccharide, as well as clear evidence that one enzyme can actually transfer two different sugars. A different enzyme from type F from P. multocida, called PMCS, was found and found to catalyze the formation of a nonsulfated polymer of chondroitin. HA and chondroitin are identical in structure, with the exception of the polymer mentioned above, which contains N-acetylglucosamine instead of GlcNAc. Both pmGCS and pmCS are 87% identical at the amino acid level. Most of the changes in residues are in the A1 region, which is consistent with the hypothesis that this region is responsible for hexosamine signaling.

Illustration 2. Schematic representation of pmGCS areas.
Two independent transferase domains, A1 and A2, are responsible for catalyzing the polymerization of the HA chain. Repeated, sequential additions of single sugars quickly build the HA chain. It appears that the carboxyl end of pmGCS interacts in some way with the membrane-bound transport machinery of the bacterial cell.

Figure 3. Model of GC biosynthesis using pmGCS.
Single sugars are added to each "A" domain in a repeated manner to the non-reducing end of the HA chain. The intrinsic precision of each step of transferase activity maintains the repetition of the HA disaccharide structure. The nascent GC chain is likely retained by pmGCs during catalysis via an oligosaccharide-binding site.

We have demonstrated efficient single-sugar signaling by pmGCS in vitro by several types of experiments, and therefore, we hypothesized that GC chains are formed by rapid, repeated addition of single sugar by class II synthase (Figure 3). To date, one line of evidence suggests that the class I enzyme also possesses two transferase sites. Mutation of leucine residue 314 to valine in mmGCS1, part of the GlcUA-tase pre-region, was reported to convert this vertebrate GCS into a chito-oligosaccharide synthase. No sites with corresponding GlcNAc transferase activity have been identified.

Polymer grafting with polysaccharide synthases: adding HA to molecules or solid particles

Research on PMGCs in the research laboratory has transformed the understanding of GC synthases from the realm of difficult, stubborn animal-like monsters to potential biotechnological workhorses. New molecules can be formed using the ability of PMGCs to graft long HA chains onto short HA-derived chains or chondroitin-derived acceptors. For example, useful scavengers may consist of small molecules or drugs with covalently linked HA or chondroitin oligosaccharide chains (4 sugars long, for example). Alternatively, HA chains can be added to an oligosaccharide primer immobilized on a solid surface (Table 4). Thus, long HA chains can be gently added to sensitive substances or delicate devices.

In another application, new chimeric polysaccharides can be formed because the use of pmGCS by the oligosaccharide acceptor is not as strict as the saccharide transferase specificity. Chondroitin and chondroitin sulfate are recognized as acceptors of PMGCs and are extended by HA chains of various lengths (Fig. 4). On the contrary, PMCS is very homologous to chondroitin synthase and recognizes and extends GC acceptors with chondroitin chains. Chimeric glycosaminoglycan molecules are formed by containing natural, specific bond compounds. These graft polysaccharides can serve to attach to a cell or tissue that binds HA to another cell or tissue that binds chondroitin or chondroitin sulfate. In certain aspects, the grafted glycosaminoglycans resemble proteoglycans, which are essential matrix components in vertebrate tissues. But since no protein linkers are present in the chimeric polymers, the antigenicity and proteolysis problems that arise around the medical use of proteoglycans are eliminated. The risk of transmission of infectious agents by tissues extracted from animals to a human patient is also reduced when using chimeric polymers.

Table 4. PMGC-initiated grafting of HA onto polyacrylamide beads. The reaction mixture contains PMGC bearing radiolabeled UDP-(14C)GlcUA and UDP-(3H)GlcNAc, as well as various immobilized sugar primers (acceptors linked by reductive amination into amino beads) were presented. The beads were washed and radioactively incorporated onto other beads, measured by liquid scintillation calculations. GC chains were grafted onto plastic beads using a suitable primer and pmGCs.

Figure 4. Schematic representation of grafted polysaccharide structures. Pasteurella spp. GC synthase or chondroitin synthase will extend certain other polymers at the non-reducing end in vitro to form new chimeric glycosaminoglycans. Some examples are depicted.

Synthesis of monodisperse HA and HA-linked oligosaccharides

In addition to adding a large polymeric HA chain to acceptor molecules, PMGCs synthesize certain smaller HA oligosaccharides ranging from 5 to 24 sugars. Using a wild-type enzyme and various reaction conditions, a HA oligosaccharide containing 4 or 5 monosaccharides extended with several sugars into longer versions, which are very often difficult to obtain in large quantities, was relatively easily prepared. We found that combining a soluble GlcUA-Tase mutant and a soluble GlcNAc-Tase mutant in the same reaction mixture allows the formation of a HA polymer if the system is equipped with an acceptor. Within 3 minutes a chain of approximately 150 sugars (-30 kDa) was made. Any single synthase mutant will not result in a HA chain. Therefore, if further control of the reaction is done by selectively combining different enzymes, UDP sugars and acceptors, then certain monodisperse oligosaccharides can be obtained (Fig. 5).

Figure 5. Preparation of certain oligosaccharides.
In this example, a GC acceptor tetrasaccharide is extended by a single chondroitin disaccharide unit using two steps with an immobilized Pasteurella species synthase mutant (shown by white arrows). The product depicted is a new hexasaccharide. Repeating the cycle one more time produces an oligosaccharide, two cycles produce a decasaccharide, etc. If the acceptor was previously conjugated to another molecule (eg a drug or drug), then the new conjugate would be extended with a short HA, chondroitin or hybrid chain as desired.

For example, in one embodiment, a mixture of UDP-GlcNAc, UDP-GlcUA and acceptor is continuously circulated through separate bioreactors with immobilized mutant synthases that transfer only a single sugar. With each bioreactor incubation cycle, a different sugar group is added to the acceptor to form small HA-specific oligosaccharides. The use of a similar pmCS mutant (eg GalNAc-Tase) in one of the steps allowed the formation of mixed oligosaccharides when using UDP-GlcNAc. The biological activity and therapeutic potential of small HA oligosaccharides is a challenging area of ​​research that will require specific, monodisperse sugars for unambiguous interpretation.

Conclusion

Apparently, there are two different classes of GC synthases. The best characterized class II enzyme from the Pasteurella species extends the HA chain by repeated addition of a single sugar to the non-reducing end of the HA chain. The direction and mode of operation of class I synthases (streptococcal, viral, and vertebrate enzymes) remain unclear. Regarding applied sciences, the ability of pmGCS to extend exogenously located acceptor molecules is useful for the design of new molecules and/or devices with potential medical applications.