Accidents with snakes of the genus Crotalus Viperidae family are less frequent, but because of the severity of the systemic response, they present high lethal potential, and the envenoming by these snakes is characterized by neurotoxic, coagulant and myotoxic action Sgrignolli et al. Crotalus durissus is a species that inhabits areas of the Central Brazilian Cerrado and arid and semi-arid regions of Northeast and fields and open areas in the south.
The subspecies Crotalus durissus terrificus is prevalent in the southeastern and southern regions Melgarejo Among the components present in the venom of snakes, those showing the greatest potential as biotechnological agents and responsible for the most serious disorders within the envenoming are the phospholipases A 2 PLA 2 , proteases, lectins type C, hyaluronidases, disintegrins and L-amino acid oxidases.
The PLA 2 s present in snake venoms are responsible for pharmacological effects involving blood incoagulability, myonecrosis, edema formation, inhibition of platelet aggregation and hypertension. These enzymes are also present in the pancreatic secretions, being widely distributed in animals and plants in nature Burke and Dennis Among the proteases present in snake venoms are the serineproteases that act selectively on factors of the coagulation cascade, with effect on platelet aggregation, fibrinolysis and coagulation Braud and Wisner and metalloproteinases, dependent on the zinc ion, responsible for bleeding, tissue damage and local inflammation Telesi and Machado In literature, little has been discussed about the venom-vitamins interaction.
Soon the investigation of the interaction between vitamins and snake venoms seems to be necessary because vitamins are molecules that are closely linked to the performance of enzymatic functions present in organisms. In addition, various enzymes, such as phospholipases and proteases present in the venoms show functional and structural homology with enzymes present in the human body, enabling similarities between the inhibition of enzymes resulting from venoms and the likely effects on human endogenous enzymes. Vitamins are the primary antioxidants obtained by feeding, being involved in processes such as cell migration, energy metabolism, vasomotor control, transportation of metal ions and glucose, amino acid formation, maturation of blood cells, cholesterol metabolism, decreased lipid membrane degradation - thereby reducing the formation of eicosanoids, repair of damage in DNA molecule, stimulation of the immune system and production of hormones You et al.
The aim of this study was to evaluate the interaction between micronutrients vitamins of B complex, vitamin E and ascorbic acid - together, and ascorbic acid alone and enzymes present in the venom of B.
Cofactor Biosynthesis: A Mechanistic Perspective
The venoms of B. The coagulant, thrombolytic, hemagglutinating and hemolytic activities were performed using human blood collected in tubes containing sodium citrate or without anticoagulant thrombolytic activity. The vitamin complex containing vitamins C, E and B complex vitamins beminal was purchased from a drugstore and the isolated vitamin C was acquired from a compounding pharmacy.
For purposes of this test, we made use of a methodology based on two methods previously described by Gutierrez et al. Thus, substrate azocasein concentration and dissolution conditions were maintained, obtaining proportions identical to those used by Van der Walt and Joubert to be incorporated into agarose gel method of Gutierrez et al. The minimum dose azocaseinolytic of venoms, responsible for the formation of an activity halo of approximately 1. For gel preparation, azocasein, at 0. The formation of translucent halo around the opening in the gel was indicative of activity, where the halos were measured in millimeters for quantifying the proteolytic activity on azocasein.
The proteolytic analysis of collagen and gelatin at 0. For displaying of results, the gel was subjected to coloring in starch black solution 0. The analyzed samples were incubated in the same proportions described for azocaseinolytic activity. Controls containing solely vitamins or venoms were also performed. The minimum coagulant dose was defined as the minimum amount of venom responsible for plasma clotting in a time interval between 1 and 1.
Then, several doses of venom were tested to determine the one responsible for a complete dissolution of the thrombus. The activity was quantified by the volume of liquid released from the thrombus, as described by Cintra et al. Controls containing only vitamins were also performed.
Ten mL of the used blood was immediately centrifuged and supernatant was discarded. The cells were suspended in saline solution 0.
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Then, the erythrocytes were suspended in the same solution 1: The results were evaluated by a qualitative visual analysis, 2 hours after addition of samples, using an electronic magnifier. The plasma was removed, and the red cells were suspended in 5 mmol L -1 phosphate buffer, pH 7. The hemoglobin concentration was determined in the supernatant by measuring the absorbance at nm Shimadzu UV 1 PC according to Rangel et al. The fibrinogenolytic activity was carried out with modifications Czaikoski et al.
The inhibitory potential of the vitamins on the proteolytic action of B. Electrophoretic run was held for a period of 2 hours at 90V. After run, gel was removed and colored for 40 minutes in 0. Statistical analyses were performed with GraphPad Prism software, and the Student's t-test was chosen to compare the experimental mediums.
All assays were performed in triplicate.
The greatest inhibitions, obtained to the proteolytic activity induced by B. Ascorbic acid and vitamin complex inhibited the azocaseinolytic activity with greater efficiency at the proportion 1: At the ratio of 1: At ratios of 1: None of the vitamin treatments proved to be effective in inhibiting proteolysis of collagen data not shown. The vitamin complex and ascorbic acid were effective in inhibiting gelatinolytic activity induced by B.
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The most effective ratio of 1: Either the preincubation of vitamin with plasma Table I and with venoms Table II , both proposed for coagulant testing, showed significant results and extended clotting time for ascorbic acid and vitamin complex. Results expressed in clotting time represent averages of triplicates and the standard deviation SD. The significance asterisks result from comparative analysis of treatments with control containing only venom. Preincubation with venom and vitamins stood out between the two treatments Table II. This suggests, therefore, possible interactions between toxins and vitamin molecules.
In addition, the largest proportions, such as 1: Hence, lower inhibitions were observed for preincubated vitamins with plasma, demonstrating non-significant changes in coagulation time induced by C. On the part of vitamins, higher proportions could not be assessed due to the saturation limit amounts of the reaction environment; however, variations in incubation time and concentration of ions can be further evaluated.
The vitamin complex preincubated with venom at 1: However, ascorbic acid showed higher inhibitory potential, being responsible for reducing the venom activity to 47, 8 and 3. This activity was completely inhibited after preincubation of the venom with ascorbic acid at the ratio of 1: Absence of hemagglutination was also observed after preincubating the venom with vitamin complex at a ratio of 1: The two treatments evaluated, ascorbic acid and vitamin complex, reduced hemolytic activity, highlighting the vitamin complex which reduced the hemolytic activity induced by the B.
Although ascorbic acid in all tested proportions has shown significant inhibition against the C. The two treatments evaluated were effective in inhibiting the degradation of fibrinogen molecules, induced by the B. Some fibrinopeptides can be seen on line 4 of Figure 5 a, which corresponds to the treatment with ascorbic acid at a ratio of 1: However, smaller and larger doses were also evaluated data not shown and shown to be without effect, only being presented the ratio of 1: As observed during caseinolytic activity tests, the inhibition differential can be related to the greater expression of proteases from snake venoms of the species B.
Therefore, the vitamin doses evaluated were not appropriate to inhibit substantially all classes of proteases expressed in the B. The lack of inhibition observed for both vitamin samples, evaluated on the proteolysis of collagen molecules, is probably due to a quick action of several proteases from the venom, which act on coagulation factors, platelets and bleeding induction, requiring additional tests to exploit the concentration-time relation in order to observe the inhibitory effect Escalante et al.
The different structures of the evaluated substrates, azocasein, gelatin and collagen, suggest the action of different proteases on each of them, enabling the interaction of these with inhibitors also in a variable form, which explains the variations in inhibition percentages observed at each test. The result is in agreement with the data obtained in coagulation assay, since the fibrinogenolysis is directly related to blood clot development Braud and Wisner Thus, the same way as vitamins proved effective in prolonging plasma-clotting time, by partially inhibiting coagulant toxins, they were also able to inhibit degradation of fibrinogen.
In the coagulant assay, the lower inhibition response by vitamin incubation with plasma can be related to the less interaction between vitamins and constituents of the plasma that participate in clot cascade, in comparison to the interactions between vitamins and molecules as PLA 2 and proteases, present in crotalic and bothropic venoms, act on coagulation.
In addition, different amounts of these molecules in the composition of venoms can interfere in the process, as well as variations in the ionic composition of the same enzyme cofactor that are able to interact with vitamins. For instance, thiol groups have been described with the ability of both chelating enzyme cofactors and interfering with enzymatic redox reactions in pharmacological tests induced by viperid venoms Sunitha et al. Another result that confirms this hypothesis is the inhibition of proteolytic activity by rosmarinic acid, cinnamic acid and their analogues Aung et al.
Thus, the aforementioned interaction seems to be one of the inhibition mechanisms performed by vitamins with antioxidant activity on the various activities induced by different proteases, such as coagulant, proteolytic on various substrates and thrombolytic activities. The inhibition of proteases, in addition to increasing vitamin survival time and decrease severity of toxic signs also contributes in preserving the cardiovascular system changes by inhibiting blood pressure lowering and depressed atrial contractility Shabbir et al.
The hemagglutinating activity exerted by C-type lectins may vary in composition of the different batches of venoms used, since this protein class has increased expression in juvenile snake specimens and semi-adults and lower expression in adults Cruz et al. Thus, the absence of activity observed for the C. Biosynthesis of Riboflavin, A. Biosynthesis of Biotin and Lipoic Acid, A.
A Perspective on Enzymatic Mechanisms, R. L-Ascorbic Acid Biosynthesis, N. Multiple Biosynthetic Pathways for Vitamin B Variations on a Central Theme, C. Biosynthesis of the Methanogenic Cofactors, R. Anmeldung Mein Konto Merkzettel 0. Modelle Anatomische Modelle Somso-Modelle. The longest running serial published by Academic Press continues its well-respected run with Volume 61 , a special volume in which a guest editor has come on board and has assembled some well-known contributors who are international authorities in the field. Together they tackle some of the latest The longest running serial published by Academic Press continues its well-respected run with Volume 61, a special volume in which a guest editor has come on board and has assembled some well-known contributors who are international authorities in the field.