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Colostrum from different mammalian species—A rich source of colostrinin
ARTICLE IN PRESS
International Dairy Journal 18 (2008) 204–209 www.elsevier.com/locate/idairyj
Colostrum from different mammalian species— A rich source of colostrinin Agata Soko"owskaa, Renata Bednarza, Magdalena Pacewicza, Jerzy A. Georgiadesb, Tadeusz Wilusza, Antoni Polanowskia, a
Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland b Georgia Cosmetics of Houston, TX, USA Received 31 January 2007; accepted 1 August 2007
Abstract ColostrininTM (CLN), a proline-rich polypeptide complex, originally isolated from ovine colostrum, has been proven to be a promising and non-toxic natural preparation capable of preventing the development of Alzheimer’s disease (AD). Utilizing a new purification protocol, colostrum from cows, goats or humans can also be used as alternative sources of this complex. Comparison of properties of preparation derived from these three species revealed no significant differences from that obtained from sheep. In aminoacid composition, as in ovine CLN, a high level of proline residues (over 20%) and acidic amino acids (approximately 18%) was found in CLN from cows, goats and humans. The comparison of the electrophoretic patterns and reversed-phase-high performance liquid chromatography (RP-HPLC) elution profiles showed the presence of additional proteins in the CLN preparations of different origin, consisting mainly of b-lactoglobulin and a-lactalbumin. These proteins could be removed by an ultrafiltration process. Colostrum, regardless of origin (ovine, bovine, caprine or human), appeared to be a rich source of CLN fractions that could be separated in gram quantities. Storage of colostrum in frozen or lyophilized form affected neither the quantity nor quality of the purified peptides compared with its preparation from fresh material. r 2007 Elsevier Ltd. All rights reserved. Keywords: Colostrum; ColostrininTM (CLN); Proline-rich polypeptide complex (PRP); Alzheimer’s disease (AD)
1. Introduction A complex of low molecular weight proline-rich polypeptides (PRPs) was first isolated from ovine colostrum in 1974 during IgG purification (Janusz, Lisowski, & Franek, 1974). Subsequent investigations on PRPs’ biological role and activity revealed the importance of various factors present in the milk-like fluid produced by the mammary gland after parturition. This first milk, colostrum, plays a basic role in the transmission of passive or active immunity from mother to neonate. Utilizing high-performance liquid chromatography (HPLC) and mass spectroscopy (MS), over 30 constituent peptides of PRP from ovine colostrum have been identified. Amino-acid analysis of the peptides present in this mixture Corresponding author. Tel.: +48 713752394; fax: +48 713752608.
E-mail address: [email protected] (A. Polanowski). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.08.004
revealed an unusually high content of proline (22%) and acidic amino acids (18%) and low percentages of alanine, glycine, arginine, methionine, and histidine, and an absence of tryptophan and cysteine residues (Kruzel, Janusz, Lisowski, Fischleigh, & Georgiades, 2001). Based on in vitro and animal studies, ovine PRP, later named ColostrininTM (CLN), was initially found to have immunomodulatory properties, and was able to induce the maturation and differentiation of murine thymocytes (Janusz & Lisowski, 1993), the production of interferon (IFN) gamma, tumor necrosis factor (TNF) alpha in human peripheral blood leukocytes and in whole blood cultures (Inglot, Janusz, & Lisowski, 1996). The experimental results strongly suggest that the PRP complex is a promising, non-toxic, natural preparation capable of preventing the development of Alzheimer’s disease (AD) (Leszek et al., 2002). A clinical study conducted on 105 patients from six psychiatric centers in
ARTICLE IN PRESS A. Soko!owska et al. / International Dairy Journal 18 (2008) 204–209
Poland showed the potential of CLN in the treatment of mild or moderate AD (Bilikiewicz & Gaus, 2004). Additionally, human clinical results correlated well with the reported positive effect of CLN on the cognitive abilities of older rats (Popik, Babula, Janusz, Lisowski, & Vetulani, 1999). Recently, 1-day-old domestic chicks were used as a model system to study the effect of colostralderived peptides on retention of memory. In a single onetrial learning paradigm, CLN demonstrated widespread efficacy as a cognitive enhancer even in animals other than mammals (Stewart & Banks, 2006). Despite therapeutic evidence, the mechanism of action of CLN in AD is still unclear and has yet to be elucidated. In the light of evidence demonstrating oxidative stress as an early event and a key factor in the pathogenesis of AD (Chauhan & Chauhan, 2006; Zhu et al., 2004), CLN’s inhibition of reactive oxygen species and nitric oxide production can result in beneficial effects in AD therapy (Bacsi, Woodberry, Kruzel, & Boldogh, 2007; Boldogh et al., 2003; Mikulska & Lisowski, 2003; Zab"ocka, Janusz, Maca"a, & Lisowski, 2005). CLN may be a promising pharmacological agent, which suppresses microglial activation by affecting the differentiation/maturation process of cells of monocyte/macrophage lineage (Kubis, Marcinkowska, Janusz, & Lisowski, 2005) and affects not only adaptive, but also innate, immunity by regulation of secretion of inflammation mediators (Zab"ocka, Janusz, Maca"a, & Lisowski, 2007). Another useful activity of CLN as a therapeutic agent is both the prevention and disruption of the amyloid beta (Ab) peptide aggregates implicated in AD (Schuster et al., 2005). The inhibition of fibril formation from Ab peptide 1–40 and its dissolution by CLN, and consequently, a reduction in the neurotoxic effects of Ab oligomer were observed. More importantly, CLN’s ability to increase neurite outgrowth in a dose-dependent manner was demonstrated in pheochromocytoma (PC12) cells (Bacsi, Stanton, Hughes, Kruzel, & Boldogh, 2005). The authors concluded that CLN induces delicate cassettes of signaling pathways common to cell proliferation and differentiation and mediates activities that are similar to those of hormones and neurotrophins, leading to neurite outgrowth. The aim of this study was to investigate the presence of the proline-rich peptide complexes in the mammalian colostrums other than ovine, i.e., bovine, caprine, and human, and show that these CLNs can serve as an alternative source of CLN fractions. The obtained CLN samples were compared with each other as well as with the original PRP complex from ovine colostrum. We sought to determine if the source of colostrum influences the quality and yield of the final CLN product. The original timeconsuming CLN purification protocol (Janusz et al., 1974), employing ion exchange, affinity, and molecular sieving chromatography combined with ammonium sulfate fractionation, was replaced by a new, two-step extraction/ purification method consisting of methanol extraction and
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ammonium sulfate precipitation as the general principle (Kruzel et al., 2004). Also, we investigated storage conditions of colostrum in a frozen or lyophilized form to compare the quality and yield of the CLN fractions with those of fresh colostrum. 2. Materials and methods 2.1. Colostrum Colostrum from two breeds of cows, black-white and red-white, collected within 24 h after calving was supplied by the University of Environmental and Life Sciences, Wroclaw, Poland. CLN from bovine colostrum was purified from fresh, lyophilized, frozen and multiply refrozen material. Ovine and caprine colostrum collected within 24 h after parturition was purchased from a local farm. Human colostrum, donated by a healthy mother was collected over 62 h starting at 18 h postpartum. The collected material from each species was pooled separately and aliquoted into 100 mL samples, frozen, and stored at 20 1C until use. 2.2. Colostrum lyophilization and rehydration Samples of 100 mL bovine colostrum were freeze-dried (Labconco Co., Kansas City, MO, USA). Each portion produced about 20 g of powder. Prior to the experiment the powdered colostrum was reconstituted in deionized water to obtain a 20% (w/v) solution and used immediately. 2.3. CLN purification The purification of the CLN fractions from the colostrum of the four species was accomplished with the new two-step extraction/purification method (Kruzel et al., 2004) shown in Fig. 1A. Briefly, methanol (100%) was stirred into the raw colostrum to a final concentration of 60% and the mixture was mechanically stirred for 10 min at room temperature. The pellet was removed by centrifugation at 15,000 g and the supernatant, containing peptides of the CLN complex, was subjected to low-temperature evaporation using a vacuum dryer (o40 1C) to remove methanol. To the remaining solution an equal volume of saturated ammonium sulfate was added to obtain a final saturation of 50%. The mixture was then rocked gently overnight at 4 1C, allowing the CLN peptides to precipitate. The pellet containing CLN fractions was recovered by centrifugation at 15,000 g 30 min, dissolved in icy distilled water, and dialyzed against water overnight (2–4 1C). 2.4. Diafiltration Diafiltration experiments were performed using a Hollow Fiber filter module (Amicon, Beverly, MA, USA; cartridge model H1P10-20, cut-off 10,000 Da). The filtrate
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Fig. 1. The purification flowchart of ColostrininTM (CLN) fractions. (A) Protocol according to Kruzel et al. (2004); (B) protocol according to the current study utilizing diafiltration. Details in Section 2.4.
velocity of 400 mL h1 was maintained by a connected peristaltic pump. Over the course of filtration the retentate volume was maintained at a nearly constant value. Diafiltration was conducted until the absorption of the filtrate at 280 nm dropped below 0.2, which was accomplished after 3 diavolumes. The filtrate was then concentrated 14-fold and washed with 3 volumes of water with a Spiral-wound module (Amicon; cartridge model S1Y1, cut-off 1000 Da). 2.5. Protein measurements and polyacrylamide gel electrophoresis Protein content was determined either by the bicinchonic acid method (Smith et al., 1985) or spectrophotometrically at 280 nm. SDS–PAGE was carried out under reducing conditions according to Scha¨gger and von Jagow (1987). The ultra-low-range molecular-weight marker (ULR MWM) calibration kit was used as a reference. Staining was done with 1% Coomassie Brilliant Blue G 250 (SigmaAldrich Co., St. Louis, MO, USA). 2.6. Amino-acid analysis Samples containing 1–3 mg of protein were treated by vapor-phase HCl hydrolysis at 112 1C for 20 h in a protein hydrolyzer (Knauer, Berlin, Germany). Hydrolyzate was derivatized manually with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (Cohen & Michaud, 1993). The derivatized amino acids were separated by HPLC on
an AccQ Tag column (160 3.9 mm ID; Waters Co., Milford, MA, USA). 2.7. Reversed-phase high-performance liquid chromatography (RP-HPLC) RP-HPLC of CLN fractions was carried out on a C-18 analytical column (10 mm Nucleosil 100 C-18, 8 mm ID, batch no. 7043; Knauer). Samples in maximal volume of 1 mL, containing about 0.5 mg of protein, were loaded onto a column equilibrated with 20% acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA). The adsorbed proteins were recovered during 60 min by gradient elution using acetonitrile (0.5% min1). Flow rate was 1 mL min1, and detection was at 220 nm. 3. Results and discussion 3.1. Comparison of CLN from colostrum of different mammalian species Separation of the PRP complex from colostrum according to the protocol described by Janusz et al. (1974) is timeconsuming and difficult to scale up. Recently, a new method for extracting and purifying CLN fractions from colostrum was developed (see Section 2) which is schematically presented as a flowchart in Fig. 1, panel A (Kruzel et al., 2004). Using this method, we found that bovine, caprine, or even human colostrum may be considered as suitable alternatives to ovine colostrum, for the purification of the PRP complex. Furthermore, the yield obtained after
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preparation from bovine colostrum was more than twice that of ovine colostrum. On a weight basis from single portions of caprine and human colostrum the amounts of CLN produced reached, respectively, about 55% and 100% of that of bovine colostrum. SDS–PAGE analysis revealed that the bovine, caprine, and human preparations consisted predominantly of peptides of molecular mass (Mr) below 14 kDa. Their electrophoretic patterns show a high degree of similarity to those of ovine CLN fractions purified by methanol extraction. However, all contained more peptides of Mr of approximately 18 kDa than the ovine PRP complex prepared by the original method, and their amounts differed among the preparations (Fig. 2). The analysis of the N-terminal amino-acid sequence (data not shown) confirmed that the contaminating protein, migrating in SDS–PAGE somewhat more slowly than the myoglobin standard (17 kDa), was b-lactoglobulin and that of 14.2 kDa was likely a-lactalbumin. It is worth noting, however, that the evidence, presented by Stewart and Banks (2006), showing positive effect of the CLN preparation obtained according to Kruzel et al. (2004) on retention of chicks’ memory, proved that its activity was not affected by contaminating proteins. The presence of 20 kDa and higher Mr proteins in human CLN preparations (Fig. 2, lane 6) presumably resulted from a change in the peptide composition of the colostrum collected for this experiment up to 62 h postpartum. This explanation is supported by observations made on ovine (Fig. 3) and bovine (data not shown) colostrums. We found that CLN purified from colostrum collected up to 24 h past parturition consisted mainly of fractions with Mr below 18 kDa, whereas in those collected during later periods of lactation, the amount of proteins of higher Mr increased, with a simultaneous reduction in the level of CLN. The analysis of the amino-acid composition (data not shown) revealed that high levels of proline residues (X20%) and acidic amino acids (ffi18%), typical for ovine
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Fig. 3. SDS–PAGE analysis of ColostrininTM (CLN) fractions purified according to Kruzel et al. (2004) from ovine colostrum collected after: (lane 1) 1 h; (lane 2) 12 h; (lane 3) 24 h; (lane 4) 36 h; (lane 5) 72 h postpartum (20 mg of protein/lane); (lane 6) ULR MWM standard.
ColostrininTM, were found to be also a characteristic feature for the bovine, caprine, and human CLN fractions. Although the CLN peptides obtained by the method depicted in Fig. 1A from the colostrums of the four different mammalian species displayed visible similarities in electrophoretic and amino-acid analyses, their RP-HPLC profiles (that from bovine colostrum is presented in Fig. 4) showed differences depending on origin of the peptides. These differences were caused by the presence of varying amounts of higher-mass peptides, whose elution from the column required a higher acetonitrile concentration which consequently influenced the elution profiles. This made their comparison difficult. A common feature of the composition of PRP complexes, regardless of the colostrum source, was the presence of a group of peptides eluting from the C18 column at acetonitrile concentrations between 30% and 40%. These peptides primarily account for the characteristic CLN fractions composition and their presence seemed to determine its activity (Kruzel et al., 2001). Therefore, it was important to seek for a method to separate the higher mass fractions. 3.2. Standardization of CLN preparations with a diafiltration process
Fig. 2. SDS–PAGE analysis of different ColostrininTM (CLN) preparations: (lane 2) CLN purified according to Janusz et al. (1974) from ovine colostrum; (lanes 3–6) CLN fractions purified according to Kruzel et al. (2004) from ovine, bovine, caprine and human colostrums, respectively (20 mg of protein/lane); (lane 1) ULR MWM standard.
After trying different cross-filtration set-ups, we experienced diafiltration as potentially most successful and decided to employ this set-up for the purification of the PRP complex. To simplify the procedure, presented in Fig. 1 panel A, we omitted the evaporation step and used alcohol extract as the starting solution. The methanol extract was diluted threefold with 50 mM formic acid before filtration to prevent aggregation of peptides. Diafiltration was conducted in a module with filter cut-off 10,000 Da until the absorption at 280 nm in the filtrate dropped below 0.2. This was achieved after about 3 diavolumes. The resulting filtrate solution was subjected to concentration with the Spiral-wound filtration system. This process is presented in Fig. 1 as flowchart B. From 1000 g of colostrum, over 120 mg of CLN polypeptide complex was
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Fig. 4. The comparison of RP-HPLC profiles of ColostrininTM (CLN) fractions purified with different protocols: (solid line) according to Janusz et al. (1974) from fresh ovine colostrum; (dashed line) according to Kruzel et al. (2004) from fresh bovine colostrum.
Moreover, it is worth noting that lyophilized colostrum may be subjected to extraction with methanol either after its rehydratation (1:5, w/v) or directly by suspending the powder in alcohol. Either way yielded similar amount of PRP complex (data not shown). Moreover, it was found that neither the yield nor the amino-acid composition of CLN fractions depended on the breed of cows (data not shown). 4. Conclusion Fig. 5. SDS–PAGE analysis of ColostrininTM (CLN) fractions purified according to Kruzel et al. (2004) from bovine colostrum diafiltrated using Hollow Fiber filtration system (cut-off 10,000 Da): (lane 2) initial feed; (lane 3) filtrate after 3 diavolumes; (lane 4) retentate after 3 diavolumes (20 mg of protein/lane); (lane 1) ULR MWM standard.
obtained. The yield appeared rather low, but since the product consisted exclusively of low-Mr peptides as illustrated in Fig. 5; the peptides with a Mr higher than 10,000 Da were eliminated, which was considered satisfactory. 3.3. Dependence of CLN quality and yield upon the storage form of colostrum The SDS–PAGE and RP-HPLC analysis showed that CLN fractions separated, regardless of the form of colostrum (fresh, frozen, multiply refrozen, and lyophilized), were very much alike and the yield of PRP fluctuated within a few percent. The results presented herein correlated well with the observation made by Playford, Macdonald, and Johnson (2000) regarding several of the commercially available colostral products that were either freeze-dried, frozen, or stored at 4 1C without detriment to their bioactivity, as determined by cell proliferation assays.
Colostrum, regardless of origin (sheep, cows, goats, humans), appeared to be a rich source of CLN fractions that could be separated in gram quantities by an alternative purification procedure based on alcohol extraction. Ready availability of bovine colostrum renders it as an ideal source for industrial production of CLN. The amino-acid compositions of these preparations were similar and displayed a high content of proline residues (X20%) and acidic amino acids (ffi18%), typical for ovine CLN. The comparison of electrophoretic patterns and RP-HPLC elution profiles of the CLN preparations obtained by the employed method revealed the presence of larger peptides with Mr above 14 kDa. These additional proteins consisting mainly of b-lactoglobulin and a-lactalbumin that could be eliminated easily by means of a diafiltration process. Additionally, analysis of the results (SDS–PAGE, RPHPLC) showed that storage of colostrum in a frozen or lyophilized form did not negatively influence either the quantity or quality of the purified CLN fractions compared with the preparation purified from fresh material. Acknowledgment This research was supported in part by a grant from ReGen Therapeutics Plc., London, UK.
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References Bacsi, A., Stanton, G. J., Hughes, T. K., Kruzel, M., & Boldogh, I. (2005). Colostrinin-driven neurite outgrowth requires p53 activation in PC12 cells. Cellular and Molecular Neurobiology, 25, 1123–1139. Bacsi, A., Woodberry, M., Kruzel, M. L., & Boldogh, I. (2007). Colostrinin delays the onset of proliferative senescence of diploid murine fibroblast cells. Neuropeptides, 41, 93–101. Bilikiewicz, A., & Gaus, W. (2004). Colostrinin (a naturally occurring, proline-rich, polypeptide mixture) in the treatment of Alzheimer’s disease. Journal of Alzheimer’s Disease, 6, 17–26. Boldogh, I., Liebenthal, D., Hughes, T. K., Juelich, T. L., Georgiades, J. A., Kruzel, M. L., et al. (2003). Modulation of 4HNE-mediated signaling by proline-rich peptides from ovine colostrum. Journal of Molecular Neuroscience, 20, 125–134. Chauhan, V., & Chauhan, A. (2006). Oxidative stress in Alzheimer’s disease. Pathophysiology, 13, 195–208. Cohen, S. A., & Michaud, D. F. (1993). Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Analytical Biochemistry, 211, 279–287. Inglot, A. D., Janusz, M., & Lisowski, J. (1996). Colostrinine: A prolinerich polypeptide from ovine colostrum is a modest cytokine inducer in human leukocytes. Archivum Immunologiae et Therapiae Experimentalis (Warsz.), 44, 215–224. Janusz, M., & Lisowski, J. (1993). Proline-rich polypeptide (PRPs)— An immunomodulatory peptide from ovine colostrum. Archivum Immunologiae et Therapiae Experimentalis (Warsz.), 41, 275–279. Janusz, M., Lisowski, J., & Franek, F. (1974). Isolation and characterization of a proline-rich polypeptide from ovine colostrum. FEBS Letters, 49, 276–279. Kruzel, M. L., Janusz, M., Lisowski, J., Fischleigh, R. V., & Georgiades, J. A. (2001). Towards an understanding of biological role of colostrinin peptides. Journal of Molecular Neuroscience, 17, 379–389. Kruzel, M. L., Polanowski, A., Wilusz, T., Soko"owska, A., Pacewicz, M., Bednarz, R., et al. (2004). The alcohol-induced conformational changes in casein micelles: A new challenge for the purification of colostrinin. Protein Journal, 23, 127–133. Kubis, A., Marcinkowska, E., Janusz, M., & Lisowski, J. (2005). Studies on the mechanism of action of a proline-rich polypeptide complex
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(PRPs): Effect on the stage of cell differentiation. Peptides, 26, 2188–2192. Leszek, J., Inglot, A. D., Janusz, M., Byczkiewicz, F., Kiejna, A., Georgiades, J., et al. (2002). Colostrinins proline-rich polypeptide complex from ovine colostrum—A long-term study of its efficacy in Alzheimer’s disease. Medical Science Monitor, 8, PI93–PI96. Mikulska, J. E., & Lisowski, J. (2003). A proline-rich polypeptide complex (PRPs) from ovine colostrum. Studies on the effect of PRPs on nitric oxide (NO) production induced by LPS in THP-1 cells. Immunopharmacology and Immunotoxicology, 25, 645–654. Playford, R. J., Macdonald, C. E., & Johnson, W. S. (2000). Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders. American Journal of Clinical Nutrition, 72, 5–14. Popik, P., Babula, B., Janusz, M., Lisowski, J., & Vetulani, J. (1999). Colostrinin, a polypeptide isolated from early milk, facilitates learning and memory in rats. Pharmacology Biochemistry and Behavior, 64, 183–189. Scha¨gger, H., & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry, 166, 368–379. Schuster, D., Rajendran, A., Hui, S. W., Nicotera, T., Srikrishnan, T., & Kruzel, M. (2005). Protective effect of colostrinin on neuroblastoma cell survival is due to reduced aggregation of b-amyloid. Neuropeptides, 39, 419–426. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., et al. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150, 76–85. Stewart, M. G., & Banks, D. (2006). Enhancement of long-term memory retention by Colostrinin in one-day-old chicks trained on a weak passive avoidance learning paradigm. Neurobiology of Learning and Memory, 86, 66–71. Zab"ocka, A., Janusz, M., Maca"a, J., & Lisowski, J. (2005). A proline-rich polypeptide complex and its nonapeptide fragment inhibit nitric oxide production induced in mice. Regulatory Peptides, 125, 35–39. Zab"ocka, A., Janusz, M., Maca"a, J., & Lisowski, J. (2007). A proline-rich polypeptide complex (PRP) isolated from ovine colostrums. Modulation of H2O2 and cytokine induction in human leukocytes. International Immunopharmacology, 7, 981–988. Zhu, X., Raina, A. K., Lee, H., Casadesus, G., Smith, M. A., & Perry, G. (2004). Oxidative stress signaling in Alzheimer’s disease. Brain Research, 1000, 32–39.
International Dairy Journal 18 (2008) 204–209 www.elsevier.com/locate/idairyj
Colostrum from different mammalian species— A rich source of colostrinin Agata Soko"owskaa, Renata Bednarza, Magdalena Pacewicza, Jerzy A. Georgiadesb, Tadeusz Wilusza, Antoni Polanowskia, a
Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland b Georgia Cosmetics of Houston, TX, USA Received 31 January 2007; accepted 1 August 2007
Abstract ColostrininTM (CLN), a proline-rich polypeptide complex, originally isolated from ovine colostrum, has been proven to be a promising and non-toxic natural preparation capable of preventing the development of Alzheimer’s disease (AD). Utilizing a new purification protocol, colostrum from cows, goats or humans can also be used as alternative sources of this complex. Comparison of properties of preparation derived from these three species revealed no significant differences from that obtained from sheep. In aminoacid composition, as in ovine CLN, a high level of proline residues (over 20%) and acidic amino acids (approximately 18%) was found in CLN from cows, goats and humans. The comparison of the electrophoretic patterns and reversed-phase-high performance liquid chromatography (RP-HPLC) elution profiles showed the presence of additional proteins in the CLN preparations of different origin, consisting mainly of b-lactoglobulin and a-lactalbumin. These proteins could be removed by an ultrafiltration process. Colostrum, regardless of origin (ovine, bovine, caprine or human), appeared to be a rich source of CLN fractions that could be separated in gram quantities. Storage of colostrum in frozen or lyophilized form affected neither the quantity nor quality of the purified peptides compared with its preparation from fresh material. r 2007 Elsevier Ltd. All rights reserved. Keywords: Colostrum; ColostrininTM (CLN); Proline-rich polypeptide complex (PRP); Alzheimer’s disease (AD)
1. Introduction A complex of low molecular weight proline-rich polypeptides (PRPs) was first isolated from ovine colostrum in 1974 during IgG purification (Janusz, Lisowski, & Franek, 1974). Subsequent investigations on PRPs’ biological role and activity revealed the importance of various factors present in the milk-like fluid produced by the mammary gland after parturition. This first milk, colostrum, plays a basic role in the transmission of passive or active immunity from mother to neonate. Utilizing high-performance liquid chromatography (HPLC) and mass spectroscopy (MS), over 30 constituent peptides of PRP from ovine colostrum have been identified. Amino-acid analysis of the peptides present in this mixture Corresponding author. Tel.: +48 713752394; fax: +48 713752608.
E-mail address: [email protected] (A. Polanowski). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.08.004
revealed an unusually high content of proline (22%) and acidic amino acids (18%) and low percentages of alanine, glycine, arginine, methionine, and histidine, and an absence of tryptophan and cysteine residues (Kruzel, Janusz, Lisowski, Fischleigh, & Georgiades, 2001). Based on in vitro and animal studies, ovine PRP, later named ColostrininTM (CLN), was initially found to have immunomodulatory properties, and was able to induce the maturation and differentiation of murine thymocytes (Janusz & Lisowski, 1993), the production of interferon (IFN) gamma, tumor necrosis factor (TNF) alpha in human peripheral blood leukocytes and in whole blood cultures (Inglot, Janusz, & Lisowski, 1996). The experimental results strongly suggest that the PRP complex is a promising, non-toxic, natural preparation capable of preventing the development of Alzheimer’s disease (AD) (Leszek et al., 2002). A clinical study conducted on 105 patients from six psychiatric centers in
ARTICLE IN PRESS A. Soko!owska et al. / International Dairy Journal 18 (2008) 204–209
Poland showed the potential of CLN in the treatment of mild or moderate AD (Bilikiewicz & Gaus, 2004). Additionally, human clinical results correlated well with the reported positive effect of CLN on the cognitive abilities of older rats (Popik, Babula, Janusz, Lisowski, & Vetulani, 1999). Recently, 1-day-old domestic chicks were used as a model system to study the effect of colostralderived peptides on retention of memory. In a single onetrial learning paradigm, CLN demonstrated widespread efficacy as a cognitive enhancer even in animals other than mammals (Stewart & Banks, 2006). Despite therapeutic evidence, the mechanism of action of CLN in AD is still unclear and has yet to be elucidated. In the light of evidence demonstrating oxidative stress as an early event and a key factor in the pathogenesis of AD (Chauhan & Chauhan, 2006; Zhu et al., 2004), CLN’s inhibition of reactive oxygen species and nitric oxide production can result in beneficial effects in AD therapy (Bacsi, Woodberry, Kruzel, & Boldogh, 2007; Boldogh et al., 2003; Mikulska & Lisowski, 2003; Zab"ocka, Janusz, Maca"a, & Lisowski, 2005). CLN may be a promising pharmacological agent, which suppresses microglial activation by affecting the differentiation/maturation process of cells of monocyte/macrophage lineage (Kubis, Marcinkowska, Janusz, & Lisowski, 2005) and affects not only adaptive, but also innate, immunity by regulation of secretion of inflammation mediators (Zab"ocka, Janusz, Maca"a, & Lisowski, 2007). Another useful activity of CLN as a therapeutic agent is both the prevention and disruption of the amyloid beta (Ab) peptide aggregates implicated in AD (Schuster et al., 2005). The inhibition of fibril formation from Ab peptide 1–40 and its dissolution by CLN, and consequently, a reduction in the neurotoxic effects of Ab oligomer were observed. More importantly, CLN’s ability to increase neurite outgrowth in a dose-dependent manner was demonstrated in pheochromocytoma (PC12) cells (Bacsi, Stanton, Hughes, Kruzel, & Boldogh, 2005). The authors concluded that CLN induces delicate cassettes of signaling pathways common to cell proliferation and differentiation and mediates activities that are similar to those of hormones and neurotrophins, leading to neurite outgrowth. The aim of this study was to investigate the presence of the proline-rich peptide complexes in the mammalian colostrums other than ovine, i.e., bovine, caprine, and human, and show that these CLNs can serve as an alternative source of CLN fractions. The obtained CLN samples were compared with each other as well as with the original PRP complex from ovine colostrum. We sought to determine if the source of colostrum influences the quality and yield of the final CLN product. The original timeconsuming CLN purification protocol (Janusz et al., 1974), employing ion exchange, affinity, and molecular sieving chromatography combined with ammonium sulfate fractionation, was replaced by a new, two-step extraction/ purification method consisting of methanol extraction and
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ammonium sulfate precipitation as the general principle (Kruzel et al., 2004). Also, we investigated storage conditions of colostrum in a frozen or lyophilized form to compare the quality and yield of the CLN fractions with those of fresh colostrum. 2. Materials and methods 2.1. Colostrum Colostrum from two breeds of cows, black-white and red-white, collected within 24 h after calving was supplied by the University of Environmental and Life Sciences, Wroclaw, Poland. CLN from bovine colostrum was purified from fresh, lyophilized, frozen and multiply refrozen material. Ovine and caprine colostrum collected within 24 h after parturition was purchased from a local farm. Human colostrum, donated by a healthy mother was collected over 62 h starting at 18 h postpartum. The collected material from each species was pooled separately and aliquoted into 100 mL samples, frozen, and stored at 20 1C until use. 2.2. Colostrum lyophilization and rehydration Samples of 100 mL bovine colostrum were freeze-dried (Labconco Co., Kansas City, MO, USA). Each portion produced about 20 g of powder. Prior to the experiment the powdered colostrum was reconstituted in deionized water to obtain a 20% (w/v) solution and used immediately. 2.3. CLN purification The purification of the CLN fractions from the colostrum of the four species was accomplished with the new two-step extraction/purification method (Kruzel et al., 2004) shown in Fig. 1A. Briefly, methanol (100%) was stirred into the raw colostrum to a final concentration of 60% and the mixture was mechanically stirred for 10 min at room temperature. The pellet was removed by centrifugation at 15,000 g and the supernatant, containing peptides of the CLN complex, was subjected to low-temperature evaporation using a vacuum dryer (o40 1C) to remove methanol. To the remaining solution an equal volume of saturated ammonium sulfate was added to obtain a final saturation of 50%. The mixture was then rocked gently overnight at 4 1C, allowing the CLN peptides to precipitate. The pellet containing CLN fractions was recovered by centrifugation at 15,000 g 30 min, dissolved in icy distilled water, and dialyzed against water overnight (2–4 1C). 2.4. Diafiltration Diafiltration experiments were performed using a Hollow Fiber filter module (Amicon, Beverly, MA, USA; cartridge model H1P10-20, cut-off 10,000 Da). The filtrate
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Fig. 1. The purification flowchart of ColostrininTM (CLN) fractions. (A) Protocol according to Kruzel et al. (2004); (B) protocol according to the current study utilizing diafiltration. Details in Section 2.4.
velocity of 400 mL h1 was maintained by a connected peristaltic pump. Over the course of filtration the retentate volume was maintained at a nearly constant value. Diafiltration was conducted until the absorption of the filtrate at 280 nm dropped below 0.2, which was accomplished after 3 diavolumes. The filtrate was then concentrated 14-fold and washed with 3 volumes of water with a Spiral-wound module (Amicon; cartridge model S1Y1, cut-off 1000 Da). 2.5. Protein measurements and polyacrylamide gel electrophoresis Protein content was determined either by the bicinchonic acid method (Smith et al., 1985) or spectrophotometrically at 280 nm. SDS–PAGE was carried out under reducing conditions according to Scha¨gger and von Jagow (1987). The ultra-low-range molecular-weight marker (ULR MWM) calibration kit was used as a reference. Staining was done with 1% Coomassie Brilliant Blue G 250 (SigmaAldrich Co., St. Louis, MO, USA). 2.6. Amino-acid analysis Samples containing 1–3 mg of protein were treated by vapor-phase HCl hydrolysis at 112 1C for 20 h in a protein hydrolyzer (Knauer, Berlin, Germany). Hydrolyzate was derivatized manually with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (Cohen & Michaud, 1993). The derivatized amino acids were separated by HPLC on
an AccQ Tag column (160 3.9 mm ID; Waters Co., Milford, MA, USA). 2.7. Reversed-phase high-performance liquid chromatography (RP-HPLC) RP-HPLC of CLN fractions was carried out on a C-18 analytical column (10 mm Nucleosil 100 C-18, 8 mm ID, batch no. 7043; Knauer). Samples in maximal volume of 1 mL, containing about 0.5 mg of protein, were loaded onto a column equilibrated with 20% acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA). The adsorbed proteins were recovered during 60 min by gradient elution using acetonitrile (0.5% min1). Flow rate was 1 mL min1, and detection was at 220 nm. 3. Results and discussion 3.1. Comparison of CLN from colostrum of different mammalian species Separation of the PRP complex from colostrum according to the protocol described by Janusz et al. (1974) is timeconsuming and difficult to scale up. Recently, a new method for extracting and purifying CLN fractions from colostrum was developed (see Section 2) which is schematically presented as a flowchart in Fig. 1, panel A (Kruzel et al., 2004). Using this method, we found that bovine, caprine, or even human colostrum may be considered as suitable alternatives to ovine colostrum, for the purification of the PRP complex. Furthermore, the yield obtained after
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preparation from bovine colostrum was more than twice that of ovine colostrum. On a weight basis from single portions of caprine and human colostrum the amounts of CLN produced reached, respectively, about 55% and 100% of that of bovine colostrum. SDS–PAGE analysis revealed that the bovine, caprine, and human preparations consisted predominantly of peptides of molecular mass (Mr) below 14 kDa. Their electrophoretic patterns show a high degree of similarity to those of ovine CLN fractions purified by methanol extraction. However, all contained more peptides of Mr of approximately 18 kDa than the ovine PRP complex prepared by the original method, and their amounts differed among the preparations (Fig. 2). The analysis of the N-terminal amino-acid sequence (data not shown) confirmed that the contaminating protein, migrating in SDS–PAGE somewhat more slowly than the myoglobin standard (17 kDa), was b-lactoglobulin and that of 14.2 kDa was likely a-lactalbumin. It is worth noting, however, that the evidence, presented by Stewart and Banks (2006), showing positive effect of the CLN preparation obtained according to Kruzel et al. (2004) on retention of chicks’ memory, proved that its activity was not affected by contaminating proteins. The presence of 20 kDa and higher Mr proteins in human CLN preparations (Fig. 2, lane 6) presumably resulted from a change in the peptide composition of the colostrum collected for this experiment up to 62 h postpartum. This explanation is supported by observations made on ovine (Fig. 3) and bovine (data not shown) colostrums. We found that CLN purified from colostrum collected up to 24 h past parturition consisted mainly of fractions with Mr below 18 kDa, whereas in those collected during later periods of lactation, the amount of proteins of higher Mr increased, with a simultaneous reduction in the level of CLN. The analysis of the amino-acid composition (data not shown) revealed that high levels of proline residues (X20%) and acidic amino acids (ffi18%), typical for ovine
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Fig. 3. SDS–PAGE analysis of ColostrininTM (CLN) fractions purified according to Kruzel et al. (2004) from ovine colostrum collected after: (lane 1) 1 h; (lane 2) 12 h; (lane 3) 24 h; (lane 4) 36 h; (lane 5) 72 h postpartum (20 mg of protein/lane); (lane 6) ULR MWM standard.
ColostrininTM, were found to be also a characteristic feature for the bovine, caprine, and human CLN fractions. Although the CLN peptides obtained by the method depicted in Fig. 1A from the colostrums of the four different mammalian species displayed visible similarities in electrophoretic and amino-acid analyses, their RP-HPLC profiles (that from bovine colostrum is presented in Fig. 4) showed differences depending on origin of the peptides. These differences were caused by the presence of varying amounts of higher-mass peptides, whose elution from the column required a higher acetonitrile concentration which consequently influenced the elution profiles. This made their comparison difficult. A common feature of the composition of PRP complexes, regardless of the colostrum source, was the presence of a group of peptides eluting from the C18 column at acetonitrile concentrations between 30% and 40%. These peptides primarily account for the characteristic CLN fractions composition and their presence seemed to determine its activity (Kruzel et al., 2001). Therefore, it was important to seek for a method to separate the higher mass fractions. 3.2. Standardization of CLN preparations with a diafiltration process
Fig. 2. SDS–PAGE analysis of different ColostrininTM (CLN) preparations: (lane 2) CLN purified according to Janusz et al. (1974) from ovine colostrum; (lanes 3–6) CLN fractions purified according to Kruzel et al. (2004) from ovine, bovine, caprine and human colostrums, respectively (20 mg of protein/lane); (lane 1) ULR MWM standard.
After trying different cross-filtration set-ups, we experienced diafiltration as potentially most successful and decided to employ this set-up for the purification of the PRP complex. To simplify the procedure, presented in Fig. 1 panel A, we omitted the evaporation step and used alcohol extract as the starting solution. The methanol extract was diluted threefold with 50 mM formic acid before filtration to prevent aggregation of peptides. Diafiltration was conducted in a module with filter cut-off 10,000 Da until the absorption at 280 nm in the filtrate dropped below 0.2. This was achieved after about 3 diavolumes. The resulting filtrate solution was subjected to concentration with the Spiral-wound filtration system. This process is presented in Fig. 1 as flowchart B. From 1000 g of colostrum, over 120 mg of CLN polypeptide complex was
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Fig. 4. The comparison of RP-HPLC profiles of ColostrininTM (CLN) fractions purified with different protocols: (solid line) according to Janusz et al. (1974) from fresh ovine colostrum; (dashed line) according to Kruzel et al. (2004) from fresh bovine colostrum.
Moreover, it is worth noting that lyophilized colostrum may be subjected to extraction with methanol either after its rehydratation (1:5, w/v) or directly by suspending the powder in alcohol. Either way yielded similar amount of PRP complex (data not shown). Moreover, it was found that neither the yield nor the amino-acid composition of CLN fractions depended on the breed of cows (data not shown). 4. Conclusion Fig. 5. SDS–PAGE analysis of ColostrininTM (CLN) fractions purified according to Kruzel et al. (2004) from bovine colostrum diafiltrated using Hollow Fiber filtration system (cut-off 10,000 Da): (lane 2) initial feed; (lane 3) filtrate after 3 diavolumes; (lane 4) retentate after 3 diavolumes (20 mg of protein/lane); (lane 1) ULR MWM standard.
obtained. The yield appeared rather low, but since the product consisted exclusively of low-Mr peptides as illustrated in Fig. 5; the peptides with a Mr higher than 10,000 Da were eliminated, which was considered satisfactory. 3.3. Dependence of CLN quality and yield upon the storage form of colostrum The SDS–PAGE and RP-HPLC analysis showed that CLN fractions separated, regardless of the form of colostrum (fresh, frozen, multiply refrozen, and lyophilized), were very much alike and the yield of PRP fluctuated within a few percent. The results presented herein correlated well with the observation made by Playford, Macdonald, and Johnson (2000) regarding several of the commercially available colostral products that were either freeze-dried, frozen, or stored at 4 1C without detriment to their bioactivity, as determined by cell proliferation assays.
Colostrum, regardless of origin (sheep, cows, goats, humans), appeared to be a rich source of CLN fractions that could be separated in gram quantities by an alternative purification procedure based on alcohol extraction. Ready availability of bovine colostrum renders it as an ideal source for industrial production of CLN. The amino-acid compositions of these preparations were similar and displayed a high content of proline residues (X20%) and acidic amino acids (ffi18%), typical for ovine CLN. The comparison of electrophoretic patterns and RP-HPLC elution profiles of the CLN preparations obtained by the employed method revealed the presence of larger peptides with Mr above 14 kDa. These additional proteins consisting mainly of b-lactoglobulin and a-lactalbumin that could be eliminated easily by means of a diafiltration process. Additionally, analysis of the results (SDS–PAGE, RPHPLC) showed that storage of colostrum in a frozen or lyophilized form did not negatively influence either the quantity or quality of the purified CLN fractions compared with the preparation purified from fresh material. Acknowledgment This research was supported in part by a grant from ReGen Therapeutics Plc., London, UK.
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