CryoLetters 24, 275-280 (2003) Ó CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK
ANTIOXIDATIVE PROPERTIES OF LACTOFERRIN FROM BOVINE COLOSTRUM BEFORE AND AFTER ITS LYOPHILIZATION B.P. Sandomirsky*, S.E. Galchenko, K.S. Galchenko Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, 23 Pereyaslavska St., 61015 Kharkov, Ukraine. E-mail:
[email protected] Abstract The effect of lactoferrin (LF) derived from native, frozen and lyophilized bovine colostrum on the intensity of free-radical processes in model systems has been investigated. It was shown that LF, not depending on the source of its obtaining, is an efficient iron chelator and decreases intensity of peroxidative processes. It was established, that antioxidative properties of LF from lyophilized colostrum have remained unchanged within 12 months of dry colostrum storage under proper conditions. Keywords: bovine colostrum, lyophilization, lactoferrin, iron chelator, chemiluminescence, peroxidative processes. INTRODUCTION According to literature data LF is a secretory component of glandular epithelial cells and has been detected in some human secretions (intestine, tears, bile) and tissues (prostate, spleen and others) (1, 4, 6). LF is also found in milk and there is a large amount of it in women and bovine colostrum (2, 10, 11). This testifies to its important role in human organism. LF participates in metabolism, regulation of cell proliferation, immune response, as well as possesses antimicrobial, antiviral and many other properties (3, 4, 6, 9, 10), in particular, capability to bind iron ions (2, 6, 8). Iron ions especially Fe2+ are efficient catalysts of the generation of active oxygen forms (singlet oxygen, hydroxyl radical, etc.), the presence of those in cells results in the development of free-radical processes, as well as biomembrane lipid peroxidation (LPO) (2, 5). Bivalent iron also initiates the reaction of chain branching in LPO. These facts stipulate the necessity to remove an excessive iron out of the organism. The main part of iron being in inactive form is accumulated in ferritin and hemosiderin, and its another part is presented as easily dissociated complexes. Therefore, it can manifest its catalytic properties. This iron is accessible for chelators (2). Both synthesized preparations and natural ones are used as iron chelators in clinics (2, 5). Due to high LF content, bovine colostrum can be an efficient regulator of iron amount in human organism, and therefore, has a positive effect for diseases, characterized by the LPO activation. Long-term storage of bovine colostrum is possible both in frozen and lyophilized forms. In this connection the aim of this work was the studying of the effect of LF derived from
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bovine colostrum on the free-radical processes intensity in model systems as well as the effect of freezing and lyophilizing of colostrum on LF properties.
MATERIALS AND METHODS Bovine colostrum of the 2nd-3rd days of lactation was used in the work. LF was obtained using the following method. Bovine colostrum was centrifuged during 15 min at 3,000 rot/min. Plasm obtained was acidified with HCl to pH 4.6 and by centrifuging separated on casein preparation (sediment) and serum. After NaOH alkalization to pH 8.0 a protein fraction, comprising LF, was sedimented from serum with ammonium sulphate (from 30 to 80% saturation). Sediment was diluted in water and solution was acidified to pH 4,0 for alactalbumin sedimentation at isoelectric point. Solution was centrifuged and its pH was achieved to 7.0 (10). LF concentration was determined according to Lowry method (7). Antioxidative properties of LF were investigated with chemiluminescence method (2). For measuring chemiluminescence the chemiluminometer (1420-2, Russia) which works in the regimen of counting the photons was used. Chemiluminescence light sum was expressed in relative unit. Some model systems were used. In the first case 100 ml of bivalent iron salt (FeSO4(NH4)2SO4×6H2O) was added to the chemiluminometer cell, containing 1ml of 50 mM of sodium-phosphate buffer and 10 mg of isolated protein. The final concentrations of Fe 2+ were 5×10-2 or 5×10-3 M. In some cases the protein and iron were not added. Chemiluminescence was induced with 100 ml of hydrogen peroxide (H2O2). H2O2 final concentrations were 0.1 or 0.01%. Mannitol in the concentration of 4.5 mg/ml was a capturer of hydroxyl radicals. The second model system comprised 1ml of egg yolk suspension, obtained by diluting with distilled water in 1:10 ratio. Before the usage this suspension was diluted with sodiumphosphate buffer in the ratio of 1:15. We added 100 ml of LF solution (10 mg/ml final concentration) to the suspension. Mixture was thermostated during 5 min at 37°C. Chemiluminescence was induced by adding 100 ml of bivalent iron salt. The final concentration of Fe 2+ was 5×10-3 M. The level of protein antioxidative activity was estimated on a light sum of a rapid flash and slow one (a number of impulses for 600 s was calculated). The third model system comprised 1 ml of physiological solution, 100 ml of rat liver homogenate, 100 ml of LF solution (final concentration of protein was 10 mg/ml). Chemiluminescence was induced by adding 100 ml of bivalent iron salt or hydrogen peroxide in final concentrations of 5×10-2 M and 0.5% respectively. Liver homogenate was obtained by its homogenization in physiological solution in the 1:2 ratio. For freezing 1 litre plastic containers with colostrum were placed into a liquid nitrogen (196°C). Thawing was done on water bath at 37-40°C. Lyophilization was done with the device for lyophilization made in Constructing and Technical Bureau with Design Unit of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine. Before lyophilization the frozen in liquid nitrogen colostrum was thawed on water bath at 37-40°C, poured with 10 mm layer on the trays and frozen with 2°C/min down to -30°C. Drying was performed at residual pressure of (1.3-1.8)×10-2 Pa and final temperature of 33-35°C in the chamber. The samples were dried to residual humidity of 1.5-2.0%. Lyophilized colostrum was packed by 300 mg into gelatinous capsules and then to plastic containers and stored in refrigerator at 4°C. Rehydration of colostrum was done with distilled water. Statistical processing was done according to Student-Fisher method. 276
RESULTS Table 1 demonstrates the data on chemiluminescence intensity in the model system depending on the concentration of bivalent iron and hydrogen peroxide in the absence and presence of LF, obtained both from native and lyophilized colostrum. Table 1. Chemiluminescence light sum (relative units) depending on the composition of model system and H2O2 concentration. Model system composition . -3 5 10 Fe2+ 5.10-2 Fe2+ LF NC 5.10-3 Fe2+ + LF NC 5.10-2 Fe2+ + LF NC 5.10-3 Fe2+ + LF LC 5.10-2 Fe2+ + LF LC
H2O2 concentration, % 0.1 0.01 768±117 944±29 2846±111 2838±85 38±3 28±2 141±18 26±2 1079±56 355±19 158±19* 29±4* 1125±68* 371±22*
*-differences are not statistically significant in comparison with NC, P > 0.05. NC - native colostrum; LC - lyophilized colostrum.
With the increase of Fe2+ ions concentration chemiluminescence intensity increased by 3.7 and 3.0 times under the concentration of hydrogen peroxide of 0.1 and 0.01%, respectively. This is related to the fact that in the medium comprising Fe2+ and H2O2 the Fenton reaction takes place. Iron initiates the decay of hydrogen peroxide to hydroxyl ion (OH-) and hydroxyl radical (OH.). With an increase of iron concentration the intensity of this reaction rises, manifesting in the augmentation of chemiluminescence light sum. The presence of protein in the system without iron does not result in a chemiluminescence while adding hydrogen peroxide. When iron and protein are present in the system, light intensity significantly decreases comparing to the system without protein. This testifies that LF, added to a system, is an efficient iron chelator. In biological objects iron participates in so-called redox-cycle, when it can be oxidized many times, and then reduced due to the presence of such compounds as ascorbate, NADH, glutathione, etc. (2). Chelators turn it out of the redox-cycle and in such a way prevent LPO processes. Adding the mannitol, which is an efficient capturer of hydroxyl radicals, results in the decrease of luminescence intensity by 3.9 times (Table 2). Table 2. Mannitol effect on chemiluminescence light sum (relative units) depending on the model system composition. Model system composition 5.10-3 Fe2+ + 0.1% H2O2 5.10-3 Fe2+ + 0.1% H2O2 5.10-3Fe2+ + 0.1% H2O2+LF NC 5.10-3Fe2+ + 0.1% H2O2+ LF NC 5.10-3Fe2+ + 0.1% H2O2+ LF LC a b
Additive
Light sum
— mannitol — mannitol mannitol
387±22 100±11a 59±5 50±4b 54±62
- differences are statistically significant in comparison with the mannitol absence, P < 0.05. - differences not statistically significant in comparison with NC without mannitol, P > 0.05.
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During combined introduction of mannitol and LF into a system luminescence intensity slightly decreases in comparison with the same system without mannitol. This fact testifies that LF results in significant reduction of the level of hydroxyl radicals in the model systems. Table 3 demonstrates the data on the effect of LF, isolated from native, frozen and lyophilized colostrum, on chemiluminescence character in the model system, which comprises yolk lipoproteids. Table 3. Effect of freezing and lyophilizing of colostrum on LF ability to reduce the development of egg yolk lipoproteids free-radical oxidation. LF source Control ( without LF) Native colostrum Frozen colostrum Lyophilized colostrum
Rapid flash light sum, relative units 20616±622 1257±551 1337±631,2 1510±701,2
Slow flash light sum, relative units 213566±6762 10300±590a 10032±620a,b 12045±749a,b
a
-differences are statistically significant in comparison with the intensity chemiluminescence without LF, P < 0.05. b -differences are not statistically significant in comparison with NC, P > 0.05.
of
As this table shows, additing of the LF obtained from native or frozen colostrum results in significant light sum decrease of both a rapid flash, which reflects the quantity of free radicals while adding iron, and slow one, characterizing the capability of lipids to be involved into LPO. LF from lyophilized colostrum possesses practically the same properties. Table 4 demonstrates the data on the chemiluminescence intensity of rat liver homogenate, induced by bivalent iron or hydrogen peroxide, and also the effect of LF, obtained from native, frozen and lyophilized colostrum on these indices. Table 4. Intensity of rat liver homogenate chemiluminescence (relative units), induced with Fe2+ and 2 2, depending on LF source. LF source Without LF Native colostrum Frozen colostrum Lyophilized colostrum a b
Chemiluminescence inductor Fe2+ 2 2 1985±201 14770±1521 611±731 9125±1007a 1,2 572±66 8883±893a,b 639±711,2 9056±1095a,b
- differences are statistically significant in comparison with the LF absence, (P < 0.05). - differences are not statistically significant in comparison with NC, P > 0.05.
The results testify to the fact that LF independently on the source of obtaining manifests antioxidative effect by reducing chemiluminescence intensity, induced with both bivalent iron and hydrogen peroxide. LF antioxidative properties are manifested in a greater extent during the chemiluminescence induction with iron than hydrogen peroxide. These properties of LF have also remained unchangeable when storing lyophilized colostrum up to 12 months, packed into gelatinous capsules under the temperature of 4 C (Table 5).
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Table 5. Intensity of rat liver homogenate chemiluminescence in presence of LF obtained from lyophilized colostrum at different terms of its storage. Chemiluminescence Inductor Fe2+ 2
2
Storage terms, months Control 668±71 8987±907
3 641±52 9927±831
6 695±60 9327±975
9 679±71 10933±1035
12 685±67 9787±1065
differences are not statistically significant in comparison with the control at all storage terms, P > 0.05.
DISCUSSION The process of cells and tissues vital activity is accompanied with LPO, when there have formed the products significantly affecting physiological, metabolic and structural cell status. The most important peculiarity of LPO processes, occurring in cells and tissues, is the fact that for the initiation and branching of free-radical chains the presence of non-hem bivalent iron ions is essential. Fe2+ ions are oxidized directly by molecular O2 which results in the forming of H2O radicals. They are capable of the LPO induction and reaction with hydroperoxides. In the latter case the free radicals, initiating new oxidation chains are also formed. LPO reactions are widely spread in various cellular membrane structures such as mitochondria, lisosomes, plasma membranes, etc. As a result of LPO processes the active products are formed (hydroperoxides, aldehydes, ketons), which react with functional groups of proteins that causes the proteins polymerisation. There are special systems in the organism, regulating the level of LPO processes: tissue antioxidants, system of enzymes – superoxidedismutase, catalase, glutathionperoxidase, as well as the system of complexformers, binding such catalysts as metal ions of changed valence, as well as iron ions. However, during some pathological states the power of these systems can be manifested insufficiently. The prevention of the LPO processes initiation is especially important at such states. Introduction into the organism of LF, which is iron chelator, comprised in a large amount in bovine colostrum, can be one of the methods. Obtained results have demonstrated that LF derived from both native and lyophilized colostrum is an efficient iron chelator due to the removal of iron out of Fenton reaction, that results in the reduction of the chemiluminescence light sum of this reaction (Table 1). LF prevents the formation of hydroxyl radicals, as it is seen from the data of the Table 2, preventing the activation of peroxidative processes at their initial stage. Antioxidative properties of LF are also manifested when adding it to more complex biological systems, such as egg yolk lipoproteids or rat liver homogenate. Colostrum lyophilization has not resulted in the reduction of LF antioxidative properties, as well as storage of lyophilized colostrum under certain conditions within 12 months. Thus, according to the results, presented in the work, it should be concluded, that LF derived from bovine colostrum has been an efficient iron chelator. LF reduces the concentration of hydroxyl radicals and significantly decreases the intensity of peroxidative processes in model systems. During freezing, lyophilization and storage of lyophilized colostrum within 12 months under certain conditions no reduction of antioxidative properties of LF were noted.
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Accepted for publication 18/7/03
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