THE LEVEL OF VITAMIN D-BINDING PROTEIN (DBP) IN SERUM OF FOALS^*

Jan P. Madej
Department of Veterinary Prevention and Immunology, Department and Clinic of Obsterics, Ruminant Diseases and Animal Health Care, Wroclaw University of Environmental Sciences, Poland

ABSTRACT

Vitamin D-binding protein (DBP) facilitates removing of actin from circulation, takes part in transport of vitamin D and its derivatives and participates in immunological response. There were estimated the concentrations of DBP in serum of foals: in 36-48^th hour after birth (after intake of colostrum), in 3-4^th week of life and from weaned foals (8-9^th month of life). The samples were collected in four foaling seasons. Results indicate that the highest concentration of serum DBP was observed in the group of 3-4 weeks old foals. The serum DBP level is similar in 8-9 months old foals and adult horses. It is probable that physiological increase of serum DBP concentration in 3-4^th week of foal's life could be connected with significant decrease of colostral passive immunity acquired in the early life by colostrum ingestion. This study also provides serum DBP level in healthy foals as a  reference for further investigations of the possible use of DBP as disease biomarker.

Key words: vitamin D-binding protein, DBP, Gc globulin, foal, ELISA.

^* – doctoral thesis

INTRODUCTION

Vitamin D-Binding Protein (DBP) also known as Gc-globulin is one of the most important elements of extracellular actin scavenger system [20]. DBP takes part in rapid removing of actin from circulation (about 30 minutes) by actin transport mainly to the liver [7,16,18,20,25]. The percentage of DBP in the complexed form was found to be significantly higher in the plasma of humans deceased from burn [24]. The authors suggest that the high levels of the actin and DBP complexes in the plasma of cadavers may reflect the release of actin from autolytic cells.

DBP can also significantly enhance the chemotactic activity of C5a and C5a des Arg (C5-derived peptides), which are concerned as the most important chemotactic factors for neutrophils, monocytes and fibroblasts [5,6,26,35]. C5a and its derivatives induce gathering of innate immunological response cells in a place of inflammatory process and stimulate activation of complement. These cells facilitate removing of necrotic cells and in consequence restoration of the tissue. It was proved that DBP level depends on extend of injury and rate of repairing processes [3].

DBP has also ability to bind vitamin D and its derivatives such 25(OH)D₃ and 1.25(OH)₂D₃ [14]. It transports cholecalciferol to the liver and then as 25(OH)D₃ to the kidney. In the kidney 25-hydroxycholecalciferol is hydroxylated to 1,25-dihydroxycholecalciferol which stimulates calcium reabsorption from the intestine. Furthermore, human and bovine DBP have been shown to bind monounsaturated and saturated fatty acids, mainly oleic and palmitic acids. By contrast, polyunsaturated fatty acids represented a minor component [8]. It is also known that DBP treated with beta-galactosidase of B cells and Neu-1 neuraminidase of T cells is converted to potent macrophage activating factor (DBP-MAF) [33].

DBP is a conservative protein, because significant part of its protein chain remains unchanged during evolution. Such structural conservatism indicates that such protein is phylogenetically very important. Homology of DBP between various animal species was discussed in other works [21,34].

In previous studies [22] we demonstrated that 36-48 hours after delivery, the DBP concentration in mares' sera was significantly higher than in non-pregnant mares. It was also proved that DBP level in mother-mares sera had no relation to age or number of deliveries.

Other studies concerned influence of DBP level in mare's serum on level of this protein in colostrum and in serum of its progeny just after delivery (unpublished data). It arises a question whether serum DBP level in the newborn depends on mother's age, number of deliveries experienced in the past, month of delivery and whether feeding with colostrum affected DBP level in foal serum.

There are in literature a lot of factors which influence serum DBP concentration like: high oestrogen status [10], pregnancy [18,32], fulminant hepatic failure [30], hepatic encephalopathy [29], liver cirrhosis [2], nephrotic syndrome [18,32], septic shock [18], trauma or injury [3,4]. In humans  DPB is a useful marker for severity of trauma. Admission levels of DBP in trauma patients predict the chance of survival [3,4,31].

According to the author knowledge there is no information in current literature about serum DBP level in foals in particular stages of development. The aim of this study was the analysis of serum DBP level changes in foals' early after colostrum ingestion (36-48 hour of life), in 3-4^th week of life, in 8-9^th month of life (after weaning) and in adult horses. These periods of foal's life were chosen because of the following reasons. In 36-48^th hour the foals have already consumed colostrum and transfer of colostrum immunoglobulins has been finished. The literature mentioned that in 3-4^th week the foals have the lower γ-globulin level [23]. Serum samples collected from weaned 8-9 months old foals inform about current condition of foal. This condition is a result of self-synthesis of immunoglobulins without influence of maternal passive immunity. At the same time it is not observed presence of colostrums antibodies, hence all proteins present in serum derive from one's own synthesis. The 3-4^th week of foal's life is of particular interest, because of the weakest immunity of foals which could threats their normal development. It is hypothesized that serum DBP level of horses could be a marker of clinically undetectable infections, trauma or other processes proceeding cell loss. The present study provides physiological serum DBP concentrations and thus could be a point of reference for further clinical applications.

MATERIAL AND METHODS

Venous blood sera were collected from 67 thoroughbred foals. The study was conducted in one stud, during four foaling seasons. In the course of the studies the foals consumed colostrum/milk and were fed with oat's grain and hay.This situation assured that all collected results are comparable, because influence of environmental factors which could disturb observations was similar to all individuals. Besides, four-year observation enables getting to know how variable environmental conditions can influence on DBP level in particular seasons. In winter the diet was supplemented with carrots, wheat bran and maize. In a term from April till October, they grazed. In the course of the studies the foals remained under continuous veterinary care and clinically manifested no pathological sings.

Blood of foals was sampled in 36-48^th hour, 3-4^th week and 8-9^th month of life, from the external jugular vein.

The blood was allowed to clot at room temperature and was centrifuged at 1200xg for 10 min. The obtained clear serum was portioned and stored at the temperature of –20ºC. Total protein concentration (TPC) in sera was estimated by biuret technique [11,27].

Furthermore, it was sampled venous blood sera of 6 mares which have not taken part in reproduction and 3 geldings of the Wielkopolska breed, 7-14 years old horses. Equal-volumed pooled horse sera derived from blood of mentioned above horses were used as a standard in enzyme-linked immunosorbent assay (ELISA).

Concentrations of DBP in sera were estimated using a self-designed ELISA, taking advantage of the cross-reaction between obtained by us rabbit or goat polyclonal antibodies (IgG) directed against human DBP and horse DBP [22]. Anti-human DBP IgG were obtained through the hyperimmunisation of goat and rabbits by commercial human DBP (G-C Globulin, MP Biomedicals Inc., Solon, OH, USA) (detailed procedure was described in 22). ELISA-plates (F96-MaxiSorp, Nunc GmBH, Wiesbaden, Germany) were coated by rabbit IgG anti-human DBP antibodies in 0,1M carbonic acid buffer pH 9.6 and incubated overnight at 4°C. After each step wells were washed 3 times with PBS-Tween 20 (0.1% v/v). Unbound sites were blocked by casein from bovine milk 1 hour at 37°C. Then tested samples diluted in 0.1% casein in PBS were put on and incubated 1 hour at 37°C. As a standard was used horse serum pool in 3 appropriate dilutions. At next step it was added second antibody (biotinylated goat IgG anti-DBP) and incubated 1 hour at 37°C. Than it was added streptavidin conjugated to horseradish peroxidase (ExtrAvidin-HRP, Sigma-Aldrich, St. Louis, MO, USA) at 1:1000 dilution and incubated 1 hour at 37°C. As a substrate it was used o-phenylenodiamine (0.5 g·L^-1) in 0.05M citrate buffer pH 5.0. Reaction was stopped after 5 min. by addition of 1M sulphuric acid. Absorbance was measured by BioTek EL340 spectrophotometer (Winooski, Vermont, USA) by wave length λ490 nm. Data were acquired and computed by use of KC3 software (BioTek Instruments, Winooski, VT, USA).

Due to the lack of purified horse DBP we used pooled horse sera as standard and results were expressed using arbitrary units – equine unit [EqU] in which:

1EqU = amount of DBP in 1 · 10^-10 L horse serum pool.

Because of different protein concentration in each sample, the obtained values of DBP, expressed in EqU · L^-1 were divided by total protein concentration in g per liter [g · L^-1] obtaining DBP/TPC coefficient in [EqU · g^-1].

The results obtained in the ELISA test were subjected to statistical analysis using Statistica 7.1 software (StatSoft Polska Sp. z o.o., Kraków, Poland). Significance of differences between the obtained results were appraised using non-parametric tests (in cases of related variables: the Wilcoxon Signed-Rank test, in cases of unrelated variables: the Mann-Whitney U test and the Kruskal-Wallis test). Relationships between variables in individual groups were determined using Spearman's rank correlation coefficient. It was assumed that differences between groups are statistically significant when p < α=0.05, where: p – the probability of obtaining a result at least as extreme as the one that was actually observed, given that the null hypothesis is true; α – significance level; N – number of cases.

RESULTS

Results of comparison of the serum DBP level in foals after transfer of colostrum IgG (36-48h after delivery) and in 3-4 weeks old foals were presented in Fig. 1. Using the Wilcoxon test, serum DBP level of foals in 36-48^th hour (mean ± standard deviation; 0.967 ± 0.352) was demonstrated to be significantly lower than in 3-4^th week of life (1.295 ± 0.384) (p=0.000011 < α=0.05; N=44).

Comparison of the serum DBP level in 3-4 weeks and 8-9 months old foals (weaned foals) was presented in Fig. 2. Using the test of Wilcoxon, serum DBP level of foals in 3-4^th week (1.461 ± 0.408) was demonstrated to be significantly higher than in 8-9^th month of life (0.788 ± 0.146) (p=0.00768 < α=0.05; N=9).

Results of comparison of the serum DBP level in weaned 8-9 months old foals and in adult horses were presented in Fig. 3. Using the Mann-Whitney U test no significant differences were disclosed in serum DBP level in 8-9 months old foals (0.828 ± 0.186) and adult horses (0.705 ± 0.082) (p=0.0864 > α=0.05; n weaned foals = 10, n adults = 9).

There were observed changes of total protein and DBP concentrations in foals' sera during four foaling seasons (Table 1). It was noticed that in second season the serum TPC in foals was lower but DBP concentration was higher than in other seasons. It was also observed that DBP concentration in adult horses shows considerably lower variation than in foals.

DISCUSSION

Comparison of serum DBP level in the foal before and after feeding with colostrum revealed that colostrum consumption had no effect on DBP level in foals (data in preparation to press). The results of previously published studies revealed also, that 36-48 hours after delivery it was observed higher serum DBP level in comparison to non-pregnant mares [22].

The serum DBP level in foals 36-48 hours after birth is almost 40% higher than in adult horses (Table 1 and 2). In young organisms rebuilding processes of several tissues are very frequent. During this processes some cells die on the way of apoptosis. Besides, immunological system in neonatal period is just adapted to new environmental conditions, which let the presence of small inflammatory processes. It is probable that above mentioned processes may cause actin release to the extracellular space. DBP as a part in extracellular actin scavenger system, severs actin filaments, caps the barbed end and prevents formation of actin filaments [13,18,20]. Increased release of actin could activate compensatory mechanism which causes greater supply of DBP in foals during this period.

Comparison of serum DBP level in 36-48 hours and 3-4 weeks old foals (Fig. 1) revealed  statistically significant increase of this parameter. It probably derived from intensive own synthesis of DBP by foals organism. Half-life of free DBP in plasma, according to different authors, is 12-24h [18]; 15h [29]; 17.2h [9] or 2-3 days [12,28]. Taking this information into consideration it could be assumed that observed serum DBP level reflects synthesis or secretion of this protein in last hours or days. It is interesting that serum DBP level in 3-4 weeks old foals is approximately 80% higher than in adult horses and over 30% higher than in 36-48 hour of life (on the basis of data from Table 1 and Table 2). In this period of foal's life is observed the most serious decrease of serum immunoglobulin level derived from half-life period of colostrums antibodies and just developing self-synthesis of immunoglobulins [15]. The lowest levels of serum immunoglobulins in foals were noticed in IgM 6-16^th day, IgA 16-24^th day of life, but IgG and IgG(T) 1-2 months after delivery [23]. Third-fourth week after delivery is a crucial period for immune-system of foal, which must manage with pressure of environmental antigens. In this period it takes a place recognizing of pathogens, creation of immunocomplexes and frequent inflammatory processes which could be manifested by increase of morbidity. It is possible that during this processes some tissues fall apart and release actin followed by stimulation organism to synthesis more DBP. Studies conducted in humans revealed that during septic shock the level of DBP is reduced by 50-80%, with simultaneous increase percentage of DBP : actin complexes from 8% (control) to 51% [17]. Septic shock involves increased generation of eicosanoids from arachidonic acid. Actin which is released in this process too, has higher affinity to DBP than arachidonic acid, forces it out and form complexes DBP : actin. This activity increases the level of free arachidonic acid which is a substrate to production of eicosanoids [17]. Moreover, because of DBP ability to binding endotoxins, it is suggested that DBP could immediately take part in removing of this substances from organism [1].

In presented studies it was compared the serum DBP level in 3-4 weeks old foals and in weanlings (8-9 months old foals) (Fig. 2). Statistical analysis revealed that serum DBP concentration in weanlings is significantly lower than in 3-4 weeks old foals. If we compare magnitude DBP/TPC ratio in serum of 8-9 months old foals and adult horses beside reproductive period, we state that this two ratios are similar (Fig. 3). Statistical analysis confirm this remark, so we can assume that 8-9 months old foals achieve level of serum DBP comparable with adult horses.

In presented studies it was observed changes of DBP level in foals' sera during four foaling seasons. Achieved data presented in Table 1 indicate that serum DBP has different concentrations in each season. The reason of such high variation could be decrease of feed quality collected in previous vegetation season. It is possible that the higher increase of serum DBP level observed in 2^nd foaling season is caused by deterioration of feed quality.

Presented studies delivered new information about serum DBP level in healthy foals during chosen stages of their development. Even thought presented results are not expressed in SI units, they are a point of reference for further investigations of the possible use of DBP as disease biomarker. The question about changing of DBP level in foal's serum during breakdown processes and measurement importance of this parameter in diagnostics, up to now has been left without answer and requires further investigation.

CONCLUSIONS

1. The highest mean level of DBP was showed in 3-4 weeks old foals, thus at the time of significant decrease of passive colostral immunity.

2. Weaned, 8-9 months old foals presented the level of DBP similar to adult horses.

3. Serum DBP level in foals revealed considerable seasonal and also interindividual variations.

4. This study provides serum DBP level in healthy foals as a reference for further investigations.

ACKNOWLEDGMENTS

The study has been supported in 2006-2007 by the grant No. 2P06K 03030.

REFERENCES

Accepted for print: 19.12.2008

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