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UDC 615.254.7:577.112
IDENTIFICATION OF BIOACTIVE SUBSTANCES OF A NEW DRUG FOR THE TREATMENT OF KIDNEY STONE DISEASE
Altai State Medical University, Barnaul
A.Yu. Zharikov, G.V. Zharikova, O.N. Mazko, O.G. Makarova, V.O. Kiryakova
Research objective - to identify and determine the mass fraction of amino acids in the composition of the new drug for kidney stone disease treatment. The identification of amino acids was carried by means of capillary electrophoresis method with the use of capillary electrophoresis system «KAPEL®-105/105M» with positive polarity of high voltage.
As a result of the conducted experiment in the composition of substance for the treatment of kidney stone disease there were identified 12 amino acids: lysine (0,2333%), tyrosine (0,1303%), phenylalanine (0,1605%), leucine+isoleucine (0,2928%), methionine (0,1928%), valine (0,1244%), proline (0,2058%), threonine (0,2081%), serine (0,3469%), alanine (0,3586%), glycine (0,4278%). The obtained results allow to assume, that these amino acids in composition of oligopeptides or in a free state, are able to determine the antilithogenic activity of the new substance of tissues of pig kidneys.
Key words: kidney stone disease, pharmacological drug, amino acids.
Introduction
Kidney stone disease (KSD) is a widespread pathology of the urinary system suffered by 10-15% of population [1]. Therefore, the overall absolute numbers of morbidity in Russia range vary up to 14 mln people. In this respect, the basic contingent of patients is represented by most productive and socially active people: men in the age of 30-50 years [1]. Against this background, the prevention and treatment of KDS is an important medical problem.
Irrespective of all the successes of modern urology, the problem of an effective and safe therapy of KDS stays currently topical. The treatment has still to be conducted mainly by means of lithotripsy of various types, especially in case of the oxalate form of KDS [2]. At the same time, the range of pharmacological products is quite restricted, and the implemented ones are not lacking in serious drawbacks [1]. Thus, the problem of search of new effective and safe medical drugs for the treatment of KDS remains a critical issue.
In terms of preclinical study, we had earlier demonstrated a high antilithogenic activity of the biomedical substance of the tissue of pig kidneys, developed in our laboratory [3]. The experiment had shown, that the three-week intake of the new biomedical substance of pig kidneys by the experimental nephrolithiasis is accompanied by a significant alleviation of the supersaturation of the urine in oxalate ions, elimination of the disturbing factor, suppression of the oxidative stress and considerable reduction of the kidney stones' number and size. The obtained results definitely raised a question of a possible mechanism(s) of antilithogenic activity of the new substance. Considering the natural origin of the new drug, it was reasonable to begin solvation of the stated problem from the identification of biologically active substances being present in it.
Research objective
The current research objective is to identify the mass fraction of amino acids in the composition of the new drug for KDS treatment.
Materials and methods
The identification of amino acids was carried by means of capillary electrophoresis method with the use of capillary electrophoresis system "KAPEL®-105/105M" with positive polarity of high voltage.
The sample preparation was carried in the following way. For the hydrolysis of proteins and peptides of the tissue material of pig kidneys to amino acids the sample weight of the pharmacological substance weighing 100 mg was placed into the hydrolysis vial, and there were added 10 ml of 6M hydrochloric acid. The hydrolysis took place at 110 Co during 16 hours. After that the sample was cooled room temperature and filtered through the filter with the 0,22 mkm pore size. The 50 mkl of substance were picked from the obtained hydrolysate, then placed into a glass weighing bottle with 10-15 ml capacity and evaporated to dryness in the stream of warm air. Further there were added 150 mkl of 0,1M sodium carbonate solution and 300 mkl of phenylisothiocyanate in isopropyl alcohol and left for 35 minutes at room temperature. Then the obtained solution was evaporated to dryness in the stream of warm air, the dry sediment was dissolved in 0,5 ml of distilled water and centrifuged at 7000 r/min during 1 minute. The supernatant was used for electrophoresis.
The electrophoresis was carried with the use of capillary with parameters Leff/Ltot=65/75 sm, ID=50 mkm by the 30Co capillary temperature, +25 kV voltage and 300 mbar/sec sample input rate. The detection was conducted by 200nm wave length. The calculation was made by means of absolute calibration method using standard amino acid solutions of various concentration.
Results and discussion
As a result of the conducted experiment in the hydrochloric hydrolysate of the new pharmacological substance for the treatment of kidney stone disease there were identified 12 amino acids: lysine, tyrosine, phenylalanine, leucine, isoleucine, methionine, valine, proline, threonine, serine, alanine, glycine (Fig. 1).
indirectly. Obviously, the results of the current experiment do not allow to determine the protein and/or peptide structure containing these amino acids. However, the mere fact of amino acid existence in the substance of the developed pharmacological drug allows to make certain assumptions of the character of its influence on the course of nephrolithiasis.
mm
Figure 1.
Electrophoregram of the new pharmacological drug for kidney stone disease
treatment
As it follows from the figure, lysine amino acid was the first to be registered on the electrophoregram, its retention time constituted 10,477 min. The retention time of the following tyrosine and phenylalanine was 10,890 and 11,062 min respectively. Further there was registered a doubled peak corresponding to leucine and isoleucine amino acids (tret.=11,900 min). After that the following amino acids were registered in chronological order: methionine (tret.= 12,183 min), valine (tret.= 12,362 min), proline (tret.= 12,637 min), threonine (tret.= 12,835 min), serine (tret.= 13,350 min), alanine (tret.= 13,525 min) and glycine (tret.= 14,438 min).
Moreover, there were determined quantitative characteristics of the identified amino acids, presented in Table 1. It turned out that the sum total of amino acids constituted 2,6813% of the studied substance sample weight.
Consequently, the research showed, that the structure of proteins and peptides of the substance of tissues of pig kidneys includes at least 12 amino acids, the number of which are characterized by explicit biological activity, which might influence the development of nephrolithiasis directly or
The role of amino acids and bioactive peptides in the nephrolithiasis pathogenesis has been discussed for quite a long time. Thus, it is stated, that the patients with oxalate and phosphate forms of KDS show 50% reduction in relation to the normal excretion of serine, glycine, taurine and isoleucine amino acids with urine. Simultaneously,
Table 1
Mass fractions of identified amino acids
No. Amino acid Mass fraction (%)
1 Glycine 0,4278
2 Alanine 0,3586
3 Serine 0,3469
4 Leucine + isoleucine 0,2928
5 Lysine 0,2333
6 Threonine 0,2081
7 Proline 0,2058
8 Methionine 0,1928
9 Phenylalanine 0,1605
10 Tyrosine 0,1303
11 Valine 0,1244
Total: 2,6813
the patients with oxalate nephrolithiasis show an additional decrease of tyrosine and ornithine excretion [4]. According to another research, there was registered a 40% reduction of lysine urine level by oxalate nephrolithiasis, while the addition of this acid into the urine contributes to the stone destruction [5]. The causes of the registered phenomena and their correlation to the particular parts of nephrolithiasis pathogenesis are still unknown. Although, these data allow to reasonably assume, that the disorders of amino acid exchange play a significant role in the KDS development. With that, 5 of the above stated amino acids are identified in the structure of the developed pharmacological drug. This allows to suppose, that the stated amino acids, can make a certain antilithogenic influence in the organism.
The role of lipid peroxidation during the process of stone formation is well known [7-9]. At the same time, the participation of glutathione and glutathione peroxidase in the compensation of the oxidative stress, appearing during lithogenesis, was frequently demonstrated [3,7-9]. As it is known, glutathione is formed in the organism as a result of glycine metabolism, identified in the composition of the studied substance. It is entirely possible, that glycine occurring in the organism is metabolized into glutathione, increasing the antioxidant potential of kidney tissue.
Interesting is the potential contribution of serine in the curative effect of the developed drug. It is known, that serine is a metabolic predecessor of cysteine - an amino acid containing sulfhydryl group in its structure. It had been supposed for quite a long time, that due to the existence of such group cysteine might block kidney glycolate oxidase inhibiting the synthesis of oxalate ions being the basis for oxalate stone formation. And these data are experimentally proved [9,10]. Moreover, cysteine can be metabolized into glutathione (the role of which is described above) and taurine -substance, the deficiency of which in the urine is registered by oxalate nephrolithiasis [4].
Attention should also be paid to the fact of methionine identification - another sulfur-containing amino acid, taking part in a wide range of biochemical reactions. Thus, for example, the antioxidant properties of methionine are known. In the context of nephrolithiasis there exist experimental data concerning the fact, that the input of methionine into rats with nephrolithiasis accompanied by induced deficiency of B6 vitamin neutralizes oxidative stress and suppresses stone formation [11].
Summing up all the above stated, it should be noted, that as a result of the conducted research, there had been discovered a number of amino acids in the composition of the developed pharmacological drug for KSD treatment, which
can play a significant role in the alleviation of lithogenic processes. It cannot be excluded, that these amino acids in composition of oligopeptides or in a free state, at least partially, are able to determine the antilithogenic activity of the new substance of tissues of pig kidneys.
Conclusion
The conducted research allowed to identify 12 amino acids (lysine, tyrosine, phenylalanine, leucine, isoleucine, methionine, valine, proline, threonine, serine, alanine, glycine) in the composition of the new pharmacological drug of pig kidneys, which can take part in the development of its antilithogenic effect.
References:
1. Voshchula V.I. Kidney stone disease: etiotropic and pathogenetic treatment, prevention. Minsk: VEVER; 2006: 107-110.
2. Kapsargin F.P., Gulman M.I., Neimark A.I. Nephrolithiasis: choice of optimal treatment. Urology. 2010; 3: 26-30.
3. Zharikov A.Yu., Lampatov V.V., Bryukhanov V.M. et al. Experimental testing of the new biomedical substance of pig kidneyes for the treatment of kidney stone disease. Siberian scientific medical journal. 2015; 35 (6): 45-51.
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8. Zharikov A.Yu., Talalayeva O.S., Zverev Ya.F. et al. Role of antioxidant therapy in pharmacological correction of experimental nephrolithiasis. Nephrology. 2010; 14 (4): 53-58.
9. Sharma M., Sud A., Kaur T. et al. N-acetylcysteine with apocynin prevents hyperoxaluria-induced mitochondrial protein perturbations in nephrolithiasis. Free Radic Res. 2016; 50 (9): 1032-1044.
10. Sharma M., Kaur T., Singla S.K. Role of mitochondria and NADPH oxidase derived reactive oxygen species in hyperoxaluria induced nephrolithiasis: therapeutic intervention with combinatorial therapy of N-acetyl cysteine and Apocynin. Mitochondrion. 2016; 27: 15-24.
11. Selvam R., Ravichandran V. Restoration of tissue antioxidants and prevention of renal stone deposition in vitamin B6 deficient rats fed with vitamin E or methionine. Indian J Exp Biol. 1993; 31 (11): 882-887.
Contacts:
656038, Barnaul, Lenina Prospect, 40. Altai State Medical University. Tel.: (3852) 241859. Email: zharikov@agmu.ru
