What are the key reactions of lysine in biochemistry. How does lysine participate in nucleophilic substitution reactions. Which reagents can modify lysine residues specifically. What are the applications of lysine modifications in protein chemistry.

Nucleophilic Substitution Reactions of Lysine

Lysine, an essential amino acid with a primary amine side chain, participates in various nucleophilic substitution reactions. These reactions are crucial for understanding protein chemistry and developing methods for protein modification and analysis.

Acylation with Anhydrides

One of the fundamental reactions of lysine is acylation with anhydrides. This nucleophilic substitution reaction involves the attack of the lysine’s primary amine on the electrophilic carbonyl carbon of the anhydride.

• Reaction type: Nucleophilic substitution
• Reagent: Anhydride
• Product: Acylated lysine

The acylation of lysine can be used for various purposes, including protein labeling and altering protein properties. How does this reaction proceed? The nucleophilic amine group of lysine attacks the carbonyl carbon of the anhydride, forming a tetrahedral intermediate. This intermediate then collapses, expelling the leaving group and forming an amide bond.

Reversible Reaction with Methylmaleic Anhydride

Lysine also undergoes a reversible reaction with methylmaleic anhydride, also known as citraconic anhydride. This reaction is particularly useful in protein chemistry due to its reversible nature.

• Reagent: Methylmaleic anhydride (citraconic anhydride)
• Reaction type: Reversible nucleophilic substitution
• Application: Temporary protein modification

Why is this reaction valuable in biochemistry? The reversibility allows for temporary modification of lysine residues, which can be useful in studying protein structure and function or in developing controlled-release systems for drug delivery.

Specific Reactions with Imidate Compounds

Lysine exhibits high specificity and yield in reactions with certain imidate compounds, which are structurally similar to esters but contain an imido group instead of a carbonyl oxygen.

Reaction with Ethylacetimidate

The reaction between lysine and ethylacetimidate is a notable example of a highly specific nucleophilic substitution reaction.

• Reagent: Ethylacetimidate
• Product: Amidino group formation
• Leaving group: Ethanol

What makes this reaction unique? The high specificity and yield of this reaction make it valuable for targeted lysine modifications. The resulting amidino group contains two nitrogen atoms attached to the central carbon, which can alter the charge and reactivity of the modified lysine residue.

Guanidination of Lysine

Guanidination is another important modification of lysine residues, involving the conversion of the primary amine to a guanidino group.

Reaction with O-Methylisourea

Lysine reacts with O-methylisourea in a nucleophilic substitution reaction to form a guanidino group.

• Reagent: O-Methylisourea
• Product: Guanidinated lysine
• Leaving group: Methanol

What is the significance of guanidination? The guanidino group contains three nitrogen atoms attached to a central carbon, which can significantly alter the charge and hydrogen bonding capabilities of the modified residue. This modification is often used to study the role of lysine residues in protein function.

Aromatic Substitution Reactions of Lysine

Lysine can participate in nucleophilic aromatic substitution reactions with specific reagents, which are widely used in protein chemistry for labeling and analysis.

Reaction with FDNB and TNBS

Two important aromatic substitution reactions of lysine involve fluorodinitrobenzene (FDNB, also known as Sanger’s reagent) and trinitrobenzenesulfonate (TNBS).

• Reagents: FDNB (Sanger’s reagent) or TNBS
• Products: 2,4-DNP-lysine or TNB-lysine
• Reaction type: Nucleophilic aromatic substitution

How are these reactions used in biochemistry? These modifications are commonly employed for protein labeling and quantification of accessible lysine residues. The resulting products have distinct spectroscopic properties, allowing for easy detection and analysis.

Dansylation of Lysine

Lysine also reacts with dansyl chloride (dimethylaminonaphthalenesulfonyl chloride) in a nucleophilic substitution reaction.

• Reagent: Dansyl chloride
• Product: Dansylated lysine
• Application: Fluorescent labeling

Why is dansylation important? The dansyl group is highly fluorescent, making it an excellent tool for protein labeling and detection. This modification allows for sensitive analysis of proteins and peptides using fluorescence spectroscopy.

Imine Formation and Reductive Amination

Lysine exhibits high specificity in reactions with aldehydes, forming imines (Schiff bases) that can be further modified through reduction.

Reaction with Aldehydes

The primary amine of lysine readily reacts with aldehydes to form imines, also known as Schiff bases.

• Reagents: Aldehydes
• Product: Imine (Schiff base)
• Reaction type: Condensation

What makes this reaction important in biochemistry? Imine formation is a key step in many biological processes and is also used in various protein modification techniques. The reversible nature of this reaction allows for dynamic modifications under certain conditions.

Reductive Amination

The imines formed from lysine and aldehydes can be reduced to form stable secondary amines.

• Reducing agents: Sodium borohydride or sodium cyanoborohydride
• Product: Secondary amine
• Application: Stable protein modification

How does reductive amination differ from simple imine formation? The reduction step converts the reversible imine linkage into a stable secondary amine, providing a permanent modification of the lysine residue. This technique is widely used for protein crosslinking and immobilization.

Applications of Lysine Modifications in Protein Chemistry

The various reactions of lysine play crucial roles in numerous applications within protein chemistry and biotechnology.

Protein Labeling and Detection

Many of the lysine modifications described above are used for protein labeling and detection:

• Fluorescent labeling (e.g., dansylation)
• Colorimetric detection (e.g., TNBS reaction)
• Radioactive labeling
• Affinity tagging

How do these modifications enhance protein analysis? By attaching detectable moieties to lysine residues, researchers can track proteins, quantify their abundance, and study their interactions with other molecules.

Protein Crosslinking and Immobilization

Lysine modifications are often employed in protein crosslinking and immobilization techniques:

• Reductive amination for protein-surface attachment
• Crosslinking proteins using bifunctional reagents
• Creating protein-polymer conjugates

What are the benefits of these applications? These techniques allow for the creation of stable protein complexes, immobilized enzymes for biocatalysis, and protein-based materials with tailored properties.

Challenges and Considerations in Lysine Modifications

While lysine modifications offer numerous advantages in protein chemistry, there are several challenges and considerations to keep in mind.

Specificity and Selectivity

Achieving specific modification of lysine residues can be challenging due to:

• Multiple reactive lysines in a protein
• Competition with other nucleophilic amino acids
• pH-dependent reactivity

How can specificity be improved? Careful control of reaction conditions, use of protecting groups, and site-directed mutagenesis can help achieve more selective lysine modifications.

Impact on Protein Structure and Function

Modifying lysine residues can potentially affect protein properties:

• Alterations in charge distribution
• Changes in hydrogen bonding patterns
• Potential disruption of active sites or binding interfaces

What strategies can minimize these impacts? Careful selection of modification sites, use of reversible modifications, and thorough characterization of modified proteins are essential to ensure that desired functions are maintained.

In conclusion, the diverse reactions of lysine provide a rich toolbox for protein chemists and biochemists. From simple acylation to complex reductive aminations, these modifications enable a wide range of applications in protein analysis, engineering, and material science. Understanding the mechanisms, specificities, and potential pitfalls of these reactions is crucial for their effective use in research and biotechnology. As new techniques and applications continue to emerge, the importance of lysine chemistry in the field of protein science is likely to grow even further.

2.1.5: A5. Reactions of Lysine

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• Henry Jakubowski
• College of St. Benedict/St. John’s University

• reacts with anhydride in a nucleophilic substitution reaction (acylation).
• reacts reversibly with methylmaleic anhydride (also called citraconic anhydride) in a nucleophilic substitution reaction.
• reacts with high specificity and yield toward ethylacetimidate in a nucleophilic substitution reaction (ethylacetimidate is like ethylacetate only with a imido group replacing the carbonyl oxygen). Ethanol leaves as the amidino group forms. (has two N -i.e. din – attached to the C)

Figure: LYSINE REACTIONS 2

• reacts with O-methylisourea in a nucleophilic substitution reaction. with the expulsion of methanol to form a guanidino group (has 3 N attached to C, nidi)
• reacts with fluorodintirobenzene (FDNB or Sanger’s reagent) or trinitrobenzenesulfonate (TNBS, as we saw with the reaction with phosphatidylethanolamine) in a nucleophilic aromatic substitution reaction to form 2,4-DNP-lysine or TNB-lysine.
• reacts with Dimethylaminonapthelenesulfonylchloride (Dansyl Chloride) in a nucleophilic substitution reaction.

Figure: LYSINE REACTIONS 3

• reacts with high specificity toward aldehydes to form imines (Schiff bases), which can be reduced with sodium borohydride or cyanoborohydride to form a secondary amine.

• Prof. Henry Jakubowski (College of St. Benedict/St. John’s University)

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Reactions of Lysine



Reactions of Lysine

BIOCHEMISTRY – DR. JAKUBOWSKI

Last Update: 
02/27/16

•  reacts with anhydride in a nucleophilic substitution reaction
  (acylation).
• reacts reversibly with methylmaleic anhydride (also called
  citraconic anhydride) in a nucleophilic substitution reaction.
• reacts with high specificity and yield toward ethylacetimidate in a
  nucleophilic substitution reaction (ethylacetimidate  is like
  ethylacetate only with a imido group replacing the carbonyl oxygen).
  Ethanol leaves as the amidino group forms. (has two N -i.e. din –
  attached to the C)

Figure:  LYSINE REACTIONS 2

• reacts with O-methylisourea in a nucleophilic substitution reaction.
  with the expulsion of methanol to form a guanidino group (has 3 N
  attached to C, nidi)
• reacts with fluorodintirobenzene (FDNB or Sanger’s reagent) or
  trinitrobenzenesulfonate (TNBS, as we saw with the reaction with
  phosphatidylethanolamine) in a nucleophilic aromatic substitution
  reaction to form 2,4-DNP-lysine or TNB-lysine.
• reacts with Dimethylaminonapthelenesulfonylchloride
  (Dansyl Chloride) in a nucleophilic substitution reaction.

Figure:  LYSINE REACTIONS 3

• reacts with high specificity toward aldehydes to form imines (Schiff
  bases), which can be reduced with sodium borohydride or cyanoborohydride
  to form a secondary amine.

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Argumentation of symptomatic therapy of acute tonsillopharyngitis | Morozova S.V.

Diagnosis and treatment of acute infectious and inflammatory diseases of the pharynx is one of the main problems solved by practical otorhinolaryngology. The pharynx, located at the junction of the respiratory and digestive tracts, is subject to the action of numerous damaging factors that can disrupt the protective and adaptive reactions and the functioning of the mucous membrane and the lymphoid apparatus of the pharynx.

The reasons contributing to the occurrence of pharyngeal pathological changes are numerous and varied: exposure to an infectious agent, diseases of the gastrointestinal tract (GIT), diseases of the teeth and periodontium, smoking, adverse environmental, professional and domestic factors, diet errors, trauma.
It is important to note the medical and social significance of the problem. Thus, acute respiratory viral diseases, the annual incidence of which is up to 8 people per 1,000 population, are accompanied by inflammation in the respiratory tract affecting the pharynx [4]. A high level of morbidity leads to a large number of outpatient visits of the working population and students, an increase in the level of temporary disability both in the number of cases and in the number of days missed due to illness and care, to significant expenses for the purchase of medicines – within the family budget and significant financial losses – on a national scale.
Undoubtedly, one of the most common diseases of the pharynx is acute tonsillopharyngitis, which is confirmed by the high level of morbidity and the appeal of patients to general practitioners and otorhinolaryngologists. In the implementation of the disease, the leading role belongs to the infectious factor (viruses, bacteria, fungi) with a significant predominance of viruses (adenoviruses, rhinoviruses, coronaviruses, influenza viruses, parainfluenza viruses, Coxsackie A viruses, etc.). According to modern domestic scientific and clinical studies, which allow a clear idea of ​​the nature and sensitivity of the key bacterial pathogens of acute infectious and inflammatory diseases of the pharynx (microorganisms isolated with a clinically significant degree of contamination over 104 CFU / ml), the leading role is played by Streptococcus pyogenes. In patients with bacterial tonsillitis, the inoculation of group A β-hemolytic streptococcus (GABHS) reaches 80–100%, less often group C and G streptococci, Corynebacterium diphtheriae, Neisseria gonorrhoeae, Arcanobacterium haemolyticum are isolated [4, 7].
In the practice of a doctor, it is necessary to take into account the importance of identifying acute “streptococcal” tonsillopharyngitis, when the disease has a severe, protracted course, there are cases of systemic inflammatory response syndrome (SIRS). With tonsillopharyngitis caused by GABHS, there is a high probability of developing severe complications and life-threatening conditions, such as meningitis, pneumonia, sepsis, toxic shock syndrome, rheumatic fever, post-streptococcal glomerulonephritis, etc.
In acute “streptococcal” tonsillopharyngitis, the most likely routes of infection are contact and airborne. The reservoir is, as a rule, a sick person or a carrier; colonization of the pathogen is possible on the skin, mucous membrane of the pharynx and nasal cavity. An alimentary route of infection is possible – with improper storage of food, in particular dairy products. Predisposing factors for the onset of the disease are local and general hypothermia, a decrease in general and local immunity, and sometimes mechanical trauma to the tonsils.
The disease is characterized by a sudden onset, intense sore throat, most pronounced when swallowing, a feeling of dryness, soreness, sore throat, moderate (febrile) fever, headache. Patients note general weakness, malaise, loss of appetite, aching pain in the limbs. The “tonsillar” lymph nodes enlarge and become painful on palpation. With pharyngoscopy, a bright diffuse reddening of the mucous membrane of the pharynx is determined, changes in the palatine tonsils may correspond to one of the forms of primary (vulgar) tonsillitis: the palatine tonsils are enlarged, edematous and hyperemic (catarrhal tonsillitis), yellowish-white plaques are visible on the surface of the palatine tonsils (lacunar tonsillitis). ), festering follicles in the form of small yellowish vesicles (follicular tonsillitis).
The McIsaac scale helps to determine the degree of probability of streptococcal etiology of acute tonsillopharyngitis at the first stage of assessing the patient’s condition, based on the clinical manifestations of the disease. Analysis and quantitative (scoring) assessment of such criteria as fever, cough, condition of the cervical lymph nodes, palatine tonsils, patient’s age allow, with a high degree of probability, to assess the risk of streptococcal infection and determine the tactics of treatment and diagnostic measures [8]. At the same time, patients with infectious and inflammatory diseases of the pharynx, of course, need a multifaceted examination, including, in particular, such informative studies as bacteriological and immunological, which makes it possible to fully diagnose both the disease and the diagnosis of the patient. It should be recognized that in modern conditions, laboratory information is extremely important for high-quality clinical diagnostics [3].
The bacteriological method is the isolation of a pure culture of the pathogen and its identification, determination of its sensitivity to antibiotics and other chemotherapeutic drugs. Both the classical culture method by inoculation and express methods for identifying group A streptococci are used: immunochromatographic rapid test for GABHS antigens in throat swabs, methods for detecting pathogen antigens (enzyme-linked immunosorbent assay and direct immunofluorescence), methods for detecting pathogen DNA (polymerase chain reaction ), which are characterized by high sensitivity and absolute specificity.
Immunological examination allows diagnosing primary and secondary immunological deficiency. Secondary (acquired) immunological deficiency – damage to the initially unchanged immune system under the influence of various exogenous factors (infections, tumors, renal failure, long-term use of glucocorticosteroids, cytostatics). Primary (congenital) immunological deficiency is much less common – the inability of the body to implement one or another link of the immune response due to a genetically determined defect or impaired development of the lymphoid system in ontogenesis.
Diagnostics involves carrying out tests of the 1st level, which include the determination of the absolute and relative number of lymphocytes, T- and B-lymphocytes, the level of serum lg (IgM, IgG, IgA), phagocytic activity of leukocytes, as well as tests of the 2nd level: determination regulatory subpopulations (T-helpers, T-suppressors), interleukin-producing activity of cells, assessment of the proliferative activity of T- and B-lymphocytes in the reaction of blast transformation, killer activity of lymphocytes, identification of immune complexes, etc.
An important stage in the diagnosis of acute tonsillopharyngitis is differential diagnosis with diseases that have a similar symptom complex (infectious mononucleosis, diphtheria). Children’s respiratory tract infections (scarlet fever, measles) are accompanied by catarrhal oropharyngeal changes. Changes in the pharyngeal mucosa in diseases of the gastrointestinal tract (chronic gastritis, peptic ulcer, oral dysbacteriosis, gastroesophageal reflux disease, cholecystitis, pancreatitis) are very common [2].
Often, the initial and determining factor, subjectively regarded as a state of ill health, which forces patients to seek medical help, is the occurrence and severity of pain when swallowing, as well as soreness, perspiration, dry paroxysmal cough. Among the extremely numerous and diverse causes of sore throat (neuralgia of the glossopharyngeal nerve, spinal diseases, cardiovascular diseases (angina pectoris, myocardial infarction), thyroiditis, injuries and foreign bodies of the pharynx and esophagus, mental disorders, AIDS, oropharyngeal cancer, etc. ) tonsillopharyngitis is one of the most common etiological factors [6, 11].
The appearance of acute pain in the throat performs a signal function in case of inflammation of the pharynx, indicates the presence of a damaging factor, initiates protective-compensatory-adaptive processes in the body, therefore, in relation to acute tonsillopharyngitis, the expression that has become a popular expression is quite appropriate: “Pain is the watchdog of health.” However, with a sharp intensity and duration of pain, overstrain occurs, the patient develops a negative psycho-emotional state, he experiences painful anguish, and there is a clear motivation to eliminate pain as an extremely negative sensation. Pain disrupts the vital activity of the human body, inevitably affects the quality of life – an indicator directly related to the state of health, from the category of positive factors goes into the category of negative ones and needs to be eliminated [1, 5]. Pain sensations are characterized by a wide range of individual fluctuations. According to the severity of pain in acute infectious and inflammatory diseases of the pharynx, patients largely judge the severity of the disease and expect from the therapy, first of all, complete relief from pain.
These factors cannot be ignored when assessing the patient’s condition, developing therapeutic tactics and determining the effectiveness of the therapy. In this case, 2 main approaches are used: assessing the intensity of pain (“how much the patient suffers”) and assessing the impact of pain on well-being in general (“psychological state, quality of life”) [13]. Scales for the subjective assessment of the severity of pain by the patient at the time of the study are widely used in clinical practice: a visual analogue scale of pain, as well as a digital rating scale that has a number of advantages, consisting of 11 points, where 0 means no pain, 10 means pain that cannot be tolerated [ 15].
In the treatment of acute infectious and inflammatory diseases of the pharynx, drugs with antimicrobial, anti-inflammatory, analgesic, immunocorrective effects are currently used: antibacterial drugs, topical immunocorrective vaccine preparations, local antiseptics, decongestants, and hyposensitizing drugs [7]. In the complex of therapeutic measures for diseases of the pharynx, accompanied by pain, fever, an important role belongs to drugs that have a symptomatic therapeutic effect. Timely and adequate symptomatic therapy for acute tonsillopharyngitis is no less important than etiopathogenetic treatment, since its goals are to reduce the severity and eliminate the clinical manifestations of the disease, which have a significant negative impact on the patient’s daily life, to preserve his ability to perform household activities and social activity.
One of the most commonly used groups of drugs with analgesic, antipyretic and anti-inflammatory effects is non-steroidal anti-inflammatory drugs (NSAIDs). The appearance of pain during inflammatory processes is associated with the formation of prostaglandins at the site of inflammation, which irritate pain receptors and cause the appearance of a subjective sensation – pain. The principle of the analgesic action of NSAIDs is based on the suppression of the activity of cyclooxygenase types 1 and 2 (COX-1, COX-2) – the main enzymes necessary for the formation of prostaglandins.
A representative of this group is the drug OKI (lysine salt of ketoprofen). The drug has anti-bradykinin activity, stabilizes lysosomal membranes and delays the release of enzymes from them that contribute to tissue destruction in chronic inflammation, reduces the release of cytokines, and inhibits the activity of neutrophils. The lysine salt of Ketoprofen has anti-inflammatory, analgesic and antipyretic effects. The rapid onset of action is due to the higher solubility of the lysine salt of ketoprofen than that of unchanged ketoprofen, faster and more complete absorption of the active substance, which leads to the peak of its plasma concentration when taken orally after 15 minutes. The effect of the drug appears after 15-20 minutes. and persists for several hours, while unchanged ketoprofen reaches a maximum only after 60 minutes. after admission [12].
The OKI preparation is convenient to use, since it has various forms of release: granules for the preparation of a solution for oral administration, rectal suppositories (for the symptomatic treatment of inflammatory processes accompanied by fever and pain), and a topical solution (for the symptomatic treatment of inflammatory oropharyngeal diseases) . For oral administration, adults and children over 14 years of age, the contents of one two-volume sachet (full dose – 80 mg) should be dissolved in half a glass of drinking water and taken 3 times a day with meals. Children 6-14 years old and elderly patients are recommended to use the contents of 1/2 two-volume sachet (half the dose – 40 mg). Rectally, children aged 6–12 years are prescribed 1 suppository of OKI 60 mg 1–2 r./day; children over 12 years old – up to 3 rubles / day. Adults are prescribed 1 suppository of OKI 160 mg 2–3 rubles / day (elderly patients – no more than 2 suppositories / day). Locally, the drug is used in the form of a rinse solution, using in adults for 1 rinse 10 ml (5 injections) of an OKI solution, previously diluted with drinking water using the glass attached to the package. Adolescents over 12 years of age should use no more than 6 ml (3 injections) of the OKI solution for 1 rinse. The procedure must be carried out 2 rubles / day.
Contraindications to the use of the drug OKI are the “aspirin triad”, hypersensitivity to NSAIDs. The drug is also contraindicated for systemic use during exacerbation of peptic ulcer and ulcerative colitis, Crohn’s disease, diverticulitis, blood clotting disorders, severe renal dysfunction, in children under 6 years of age, in the third trimester of pregnancy and during lactation.
The analgesic effect of AEI is based on a triple mechanism: peripheral – through blockade of the arachidonic acid cycle, central – due to a decrease in the sensitivity of brain receptors and due to blockade of impulse transmission in the spinal cord. Comparative studies have shown that the analgesic efficacy of OCI is higher and manifests itself faster than that of ibuprofen and paracetamol [14]. The antipyretic effect of OCI is comparable to that of paracetamol and is more pronounced than that of ibuprofen and other NSAIDs [9]. According to the anti-inflammatory effect, OKI demonstrates high activity, significantly exceeding the activity of nimesulide [10]. Taking into account the fact that the use of nimesulide is limited in Europe, OKI as a lysine salt of ketoprofen is a worthy alternative to nimesulide, which minimizes the risk of developing adverse events from the liver.
An important aspect of the use of drugs for symptomatic therapy is their safety and tolerability. When prescribing NSAID therapy, special attention is paid to the undesirable effects inherent in the drugs of this group, primarily from the gastrointestinal tract and the hematopoietic system. Abdominal pain, diarrhea, exacerbation of gastritis or peptic ulcer, hepatic reactions – this is not a complete list of possible adverse events. In this aspect, the lysine salt of ketoprofen has advantages over ketoprofen, since it causes side effects much less frequently. Due to its chemical composition, OKI quickly mixes with a neutral pH environment and, due to this, has almost no irritating effect on the gastrointestinal mucosa. According to L. Bellussi et al. (1996), against the background of a 10-day intake of the OCI drug, the overall tolerability of therapy by patients and gastroscopy data were comparable with those when taking placebo [10].
To achieve the desired effect of pharmacotherapy, a patient with acute infectious and inflammatory diseases of the pharynx must follow the following basic recommendations: completely give up smoking and alcohol; exclude hot, cold, spicy, salty foods and drinks from the diet; in the absence of contraindications, conduct warming and distracting physiotherapy procedures at home.
Thus, the rational use of drugs for symptomatic therapy allows not only to achieve a positive clinical effect in the treatment of acute inflammatory diseases of the pharynx, but also to increase the level of patient satisfaction with the results of treatment.

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2. Vesyaeva A.A. Treatment of chronic pharyngitis associated with pathology of the gastrointestinal tract: Proceedings of the XVII Congress of Otorhinolaryngologists of Russia, Nizhny Novgorod, June 2006, pp. 165–166.
3. Menshikov V.V. (ed.), Dedov I.I., Makolkin V.I., Mukhin N.A. Clinical diagnosis – laboratory basis. M.: Labinform, 1997. 304 p.
4. Kolobukhina L.V. Viral infections of the respiratory tract // RMJ, 2000. V. 8. No. 13-14 (114-115). pp. 559–564.
5. Lerish R. Fundamentals of physiological surgery. Essays on the vegetative life of tissues / transl. from French B.M. Nikiforova M.: Medgiz, 1961. 292 p.
6. Morozova S.V. Pain in the throat: causes and possibilities of drug therapy // BC. 2005. V. 13. No. 21. S. 1447–1452.
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9. D’Arienzo M. Summary of product characteristics // Drugs Exptl.Clin.Res. 1984 Vol. X(12) . R. 863–866.
10. Bellussi L. et al. Antiphlogistic therapy with ketoprofen lysine salt vs nimesulide in secretory otitis media, rhinitis/rhinosinusitis, pharyngitis/tonsillitis/tracheitis // Note di Terapia Otorinolaringol. 1996 Vol. 46. ​​R. 49–57.
11. Denk-Linnet D.-M., Mancusi G. Current diagnostics of swallowing disorders: the ENT specialists and phoniatricians role // European Archives of Oto-Rhino-Laringology and Head & Neck. June 2007 Vol. 264. Suppl. 1. HIC 5. R. 6.
12. Fatti F. Summary of product characteristics. Data on file, 1991.
13. Haefeli M., Elfering A., Aebi M. et al. What constitutes a good outcome in spinal surgery? A preliminary survey among spine surgeons of the SSE and European spine patients // Eur Spine J. 2008. 17. P. 104–116.
14. Ligniere G.C. et al. Ricenti Progressi in Medicina, II Pensiero Scientifico Editore. 1996 Vol. 87 (supplemento al #3).
15. Wewers M.E., Lowe N.K. A critical review of visual analogue scales in the measurement of clinical phenomena // Res Nurs Health. 1990 Vol. 13. R. 227–236.

Faculty of Biology St. Petersburg State University