Clinical Trial on the absorption of natural Vitamin C and antioxidative capacity in horses..
The absorption of natural vitamin C in horses and antioxidative capacity: a randomised, controlled study on trotters during a three month training period.
Winther (1), A. Kharazmi (2), A. S. V. Hansen (3) and J. Falk-Rnne (4).
1) Department of Clinical Biochemistry, Frederiksberg Hospital, University of Copenhagen, Nordre, Fasanvej 57, 2000, Fredriksberg.
2) Department of Microbiology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 , Copenhagen.
3) Institute for Plant Biology and Biotechnology, University of Copenhagen, Faculty of Life Sciences, Blowsvej 17, 1870 Frederiksberg.
4) The Horseclinic at Lunden, Ordrup Jagtvej 201, 2920 Charlottenlund, Copenhagen, Denma
Received: 19 October 2011 / Accepted: 10 September 2012 2012 Wageningen Academic Publishers
The aim of the present study was to assess, whether low dose vitamin C supplemented in its natural form, as a dried powder from a selected subspecies of Rosehip (Rosa canina), would result in a detectable increase in the concentration of blood vitamin C in horses supplemented daily for three months. Sixteen horses were randomly allocated to two groups and supplemented with either 25 g rose-hip powder daily (equal to 125 mg natural Vitamin C), or with 50 gram rosehip powder daily (equal to 250 mg Vitamin C) daily. Serum Vitamin C was analysed both prior to supplementation and then again 2, 4 and 12 weeks following supplementation.
Whilst there was a trend towards an increase in serum vitamin C concentration following 12 weeks of supplementation with 125 mg of natural vitamin C, this was not statistically significant. However, a significant increase in serum Vitamin C concentration was observed following 2 weeks of supplementation with 250 mg vitamin C (p<0.02). The magnitude of this increase was greater following 4 and 12 weeks of supplementation (p<0.02 and p<0.02), respectively. Additionally, serum vitamin C concentration was significantly higher following supplementation with 250 versus 125mg of vitamin C daily (p<0.03). These results suggest that natural vitamin C from Rosehips absorbed sufficiently to raise serum vitamin C concentration in horses supplemented with less than 500mg daily.
Exercise and the associated increase in oxygen consumption and increased flux through the aerobic energy generating pathways are linked with an increased production of free radicals. When the production of free radicals overwhelms the bodys antioxidant defences, oxidative stress can occur with subsequent deleterious effect on cellular structures. In horses, oxidative stress has been shown to occur during exercise  and has also been implicated in the aetiology of a number of equine diseases including recurrent airway obstruction , exercise induced pulmonary haemorrhage , joint disease  and muscle damage . Oxidative stress is more likely to occur when dietary antioxidant provision is sub optimal, as reviewed by . Ascorbic acid is a major component of the antioxidant defences in lung lining fluid and vitamin C status has been identified as a factor in the pathogenesis of chronic inflammatory airway diseases in horses . Furthermore a significant antioxidant function for Vitamin C in synovial fluid has also been described in horses . However, unlike humans, but like most other vertebrates, the horse under normal circumstances has no dietary requirement for ascorbic acid, as it is synthesised in the liver ultimately from glucose. Vitamin C status is reported to be reduced following prolonged exercise  and dietary supplementation with vitamin C has been suggested to be a significant factor in maintaining ascorbic acid status in times of stress, or where disease is present .
Specifically it has been suggested that supplementation with Vitamin C, could moderate oxidative stress and thereby ameliorate muscle damage  inflammatory airway disease [10, 11] in horses and inflammatory joint disease in humans .
The role of vitamin C in inflammatory disease in horses is therefore reasonably well established. Vitamin C status is also reduced with infection [13, 14] and previous studies have shown an improvement antibody response to vaccine in aged horses following short term bolus administration of vitamin .
Absorption of vitamin C is reported in the horse to occur by passive diffusion in the ileum  and not by sodium dependent active transport as for humans . Absorption of vitamin C in the horse appears to be affected by both the level and form in which it is supplied. Although plasma level of vitamin C was elevated by feeding 20g of ascorbic acid daily for 25 days, it was not affected by a single 20g administration . Ascorbyl palmitate, a lipid soluble derivative of ascorbic acid, is more bioavailable than either ascorbic acid or ascorbyl stearate, although there is significant variation between individuals [19, 20]. Calcium ascorbyl 2 monophosphate also failed to increase plasma Vitamin C following 2 weeks of supplementation (20mg/kg bodyweight) daily.
In contrast, it has recently been reported that the administration of a single dose of natural vitamin C (1 gram) as a standardized powder from certain selected subspecies of Rosehip significantly increased vitamin C concentration in blood 2 hours following administration .
The present study aimed to investigate whether long term administration of a relatively low level of natural vitamin C (from 125 to 250 mg daily) would result in any detectable increase in serum vitamin C in horses and further to elucidate if such changes have any impact on oxidative stress, when estimated as the release of oxidative anions from polymorphnucleated leucocytes (PMN`s).
Materials and methods:
Sixteen horses (Standardbred trotters) 9 mares and 7 geldings, mean age 3.4 years (range 2 6 years) were all maintained on the same standardized dietary management and training programme for a period of 3 months. All of the horses used in this study were in active exercise training and undertook regular competition. All horses included in the trial were free from known respiratory disease, previous injury or behavioural issues.
Horses were randomly divided into two groups of 8 and were supplemented with either 25 g or 50 g of Rosehip powder daily. The amount of Rosehip powder fed was equivalent to 125 mg or 250 mg of vitamin C, respectively. In order to ensure that the study was carried out on a blinded basis, the group of horses giving 25g of Rosehip were also supplemented with a placebo powder of similar colour, odour and taste fed at a ratio of 1 to 1. Each horse in the study was therefore given an identical weight of powdered supplement daily. The supplement containers where randomly labelled from 1 to 16 and randomization took place in blocks of 4 yielding two groups of eight horses, one group receiving 125 mg and the other receiving 250 mg of natural vitamin C.
The Rosehip powder was top-dressed onto the standardised daily diet for a period of three months from August to October inclusive. The two groups were balanced for age of horse and level of training but not sex. There were 6 mares and 2 geldings in the high-dose group and 6 geldings and 2 mares in the low-dose group. The study was completed by all 16 horses with none being withdrawn.
The Rosehip supplement (Rose-Hip Vital, Hyben-Vital, Langeland, Denmark) is a powder, which consists of the entire rose-hip fruit (shells as well as seeds) from selected subspecies of Rosehip (Rosa-canina).
The Rosehip powder (Rose-Hip Vital) is sourced from plants that are grown according to good agricultural practice and harvesting takes place only once the fruits are mature. Immediately after harvesting, the fruits are frozen. When the fruits are thawed, laser technology is used to ensure that only high quality berries are used for processing. The Rosehips are then dried using a patented prolonged drying process where the temperature never exceeds 40oC and runs for several days. This drying methodology ensures that heat labile active ingredients are preserved. The resulting ingredient is a dry powder that contains the seeds and shells of the rosehip, but where the itchy hairs have been removed, using a further patented methodology. This Rosehip powder is standardised for vitamin C to a level of 500 mg/100g. The concentration of a further active ingredient, an anti-inflammatory agent and member of the galactolipid family (GOPO), which is present in higher amounts in very few plant species , is also standardised to a level of 150ug/g.
Blood samples for the analysis of vitamin C and a marker of oxidative stress were taken in the morning at 7am, immediately prior to feeding by venupuncture of the Jugular vein using two 5ml vacutainers. Blood samples were taken prior to supplementation and again following 2, 4 and 12 weeks of supplementation with Rosehip. Vitamin C was analysed using an established photometric methodology . The release of oxidative anions from polymorph nucleated leucocytes (PMN`s) was assessed at the start, after 2 weeks and at the end of the study after 3 months using chemiluminescens .
All data are presented as mean +/- SD. The non-parametric Wilcoxon test was used for statistical comparison within horse groups, whereas the non parametric Mann-Whitney test was used for statistical comparison between horse groups. The comparison of data between the two groups over time was performed using analysis of variance for repeated measures (ANOVA). Statistical significance was declared p at p< 0.05.
Mean pre-supplementation concentration of serum vitamin C in the 125mg group was 20.0 +/- 4.3 umol/L, which was not significantly different from that observed in the group supplemented with 250mg of Rosehip (18.5 +/- 5.5 umol/L). Mean concentration of vitamin C in serum following 14, 4 and 12 weeks of supplementation with 125g of Rosehip was 18.8 +/- 3.5 , 20.1 +/- 3.9 and 23.5 +/- 6.7 umol/l, respectively. Whilst there was a small increase in serum vitamin C concentration over the period of supplementation in the group fed 125mg of Rosehip, this was not significantly different from the baseline pre supplementation concentration. In contrast, mean serum vitamin C was significantly increased compared to the pre supplementation level in the group fed 250mg of Rosehip after 2 weeks of supplementation 21.4 +/- 5.5 vs. 18.5 +/- 5.5 umol/l (p<0.05) and was also significantly different form that observed in the group fed 125mg of Rosehip after the same time point (18.8 +/- 3.5 umol/l) (p<0.05). There was then a further, significant increase in mean serum vitamin C, in the 250mg supplemented group after 4 and 12 weeks (23.4 +/- 7.2 and 26.7 +/- 6.9 umol/l), respectively (p<0.05) reaching a concentration 40% above the initial pre supplementation level (figure 1).
Whilst the increase in vitamin C concentration in the group of horses fed 125mg of vitamin C was not significant compared to the pre supplementation level, it had increased sufficiently by 12 weeks of supplementation to ensure that there was no longer a significant difference in serum vitamin C concentration between horse groups.
The relative increase in serum vitamin C concentration above the pre supplementation level was calculated for both groups ( vitamin C, figure 2). Mean vitamin C was 5.75+/- 8.09 umol/l following 12 weeks of supplementation with 25g of Rosehip and 8.86+/- 8.35 umol/l following 50g of Rosehip, respectively. Comparison of the two groups over the period of supplementation (ANOVA) revealed a significant change in vitamin C (p<0.03, figure 2).
Figure 1. Serum vitamin C levels in each of the horses daily given (A) 25 g and (B) 50 g of standardised rosehip supplement. Mean values are given for each of the two groups of horses at the 4 different time points. The lower and upper 95% confidence intervals are given in brackets
Chemiluminescence analysis revealed that there was no significant difference in the liberation of oxidative anions between the 2 groups prior to supplementation with Rose hip (4498 +/-1390mV vs. 4304+/-1178mV, p<0.96, Table 1). There was also no significant change in the release of oxidative anions during the course of the study in the group supplemented with 125mg of Rosehip (table 1). However in contrast, a significant decline in the liberation of oxidative anions was observed after 2 weeks and 12 weeks of supplementation in the 250mg group (p<0.01). In addition, the decline in the liberation of oxidative anions observed after 2 weeks of supplementation was significantly different to the corresponding value for the 125mg group (p<0.05) when either the absolute values (p<0.01) or values were compared.
In common with the vitamin C data, comparison of the two supplementation groups after 12 weeks of supplementation, revealed no significant difference in the liberation of oxidative anions.
The pre supplementation concentration of vitamin C in plasma was similar to that previously reported . The present data reports that 250 mg of natural vitamin C daily, in the form of 50 g of a Rosehip supplement (Rose-Hip Vital), results in a significant increase in serum vitamin C level of horses, after 2 weeks. If supplementation is maintained for approximately 3 months, the magnitude of the increase in serum vitamin C is doubled to reach a level in serum of 26.7umol/l, which was approximately 30% above the initial pre supplementation level. In contrast, a lower level of supplementation with Rosehip (Rose-Hip Vital) of 25 g per day (equivalent to 125 mg of natural vitamin C), did not result in the same significant increase in serum vitamin C concentration, although some increase was observed after approximately 3 months of supplementation.
Table 1. The release of oxidative anions from polymorph-nucleated leucocytes in horses given daily rosehip supplementation, estimated as chemiluminescence (mV). Data are given as mean values, with lower and upper 95% confidence intervals between brackets
The measurement of oxidative stress in this study, estimated by measuring the liberation of oxidative anions from PMN`s shows a similar pattern. Supplementation for 2 weeks with 250mg of vitamin C from Rosehip resulted in a significant decline in oxidative stress. This change was not observed in the 125 mg group. After 12 weeks of supplementation, however, oxidative stress tended to decline, in this group also, although this did not attain statistically significance.
Based on the present data the authors suggest that 50g of Rosehip (Rose-Hip Vital) fed daily, which is equivalent to 250 mg of natural vitamin C, is needed to raise serum vitamin C concentration in horses and to modify oxidative stress.
This data should, however, be interpreted with some caution as the ability to raise serum vitamin C concentration appeared to be related to pre supplementation vitamin C status. Serum vitamin C concentration was in fact increased significantly in response to 12 weeks of supplementation with 125mg of vitamin C in a subgroup of horses with the lowest pre supplementation concentration of serum vitamin C (14.9 to 23.1 umol/L) (p<0.031). This was not apparent following 14 or 28 day of supplementation with 125mg of vitamin C. It is therefore possible, that serum vitamin C may have increased significantly in all horses in the 125 mg group had the 25 g of Rosehip been fed for more than 12 weeks.
Vitamin C is known to enter the plasma and extracellular fluid relatively quickly from the gastrointestinal tract. From this extracellular phase, vitamin C is then taken up by a variety of different cell types including leucocytes. It has been suggested that a two compartment system exists with respect to vitamin C and that serum vitamin C will only increase once certain cells have become saturated . This may explain why an increase in serum vitamin C in those horses fed the lower amount of Rosehip (Rose-Hip Vital) 25g was only achieved after 12 weeks compared to the 2 weeks in the horses supplemented with 50g of Rosehip (Rose-Hip Vital).
The present results are very interesting in the context of earlier studies using synthetic vitamin C. A single administration of 20,000mg synthetic vitamin C (ascorbic acid), given to horses, did not result in any detectable increase in blood [26, 27]. However, it has been reported that 20,000mg of synthetic vitamin C added daily to the drinking water of 6 horses, over a period of 25 days, did result in some improvement in the concentration of vitamin C in blood, although the variation between individuals was pronounced . The increase in vitamin C observed in this study in relation to 250mg ascorbic acid per horse per day (~2mg/kg BW) is comparable to that previously reported in ponies by . In the latter study, however, the level of supplementation with either calcium ascorbyl 2 monophosphate, or ascorbyl palmitate was significantly higher (20mg/kg BW). In addition, ascorbyl palmitate has been reported to offer greater bioavailability in horses compared to ascorbic acid .
This study therefore suggests that a comparable increase in serum vitamin C concentration can be achieved with this natural form of vitamin C from Rosehip (Rose-Hip Vital) using a fraction of the amount of synthetic sources previously used. The bioavailability of micronutrients such as vitamin E in horses has been previously reported to be increased when presented in a natural form .
The authors have shown in an earlier study that a single administration of 1000 mg of natural vitamin C, when fed as 210 g of Rosehip powder (Rose-Hip Vital) resulted in a significant increase in the serum concentration of vitamin C in horses after just 2 hours (p<0.05) . The present data indicate that 250 mg of natural vitamin C, when supplemented on a long term basis improves serum vitamin C status. It is interesting to note that such a low level of supplementation with natural vitamin C, given as a powder consisting of the entire dried fruit of a subtype of Rosehip, yields an increase in serum vitamin C which resembled the concentration obtained following supplementation with synthetic vitamin C as ascorbic acid at a level 80 times higher [26, 27] and that this low level of supplementation can influence the liberation of oxidative anions.
It was not the aim of the present study to explain the basic mechanisms of this phenomenon, but the authors suggest that within the matrix of the rosehip powder there may be some carriers or co-factors, which facilitate the uptake of vitamin C from the digestive tract. The relevance of this study lies in the role of vitamin C in maintaining respiratory function and joint integrity. It has been previously established the vitamin C content of lung lining fluid is influenced by the vitamin C content of the diet . Furthermore the importance of pulmonary vitamin C status in horses with RAO has also been previously reported [10, 29]. However, despite the purported role of vitamin C in the turnover and repair of cartilage, it is currently unclear whether dietary vitamin C has an impact on osteoarthritis in an ascorbate synthesising animal such as the horse  in comparison to humans where vitamin C is not endogenously synthesised and where it has been reported to have benefit in prevention of osteoarthritis . The provision of natural vitamin C in its natural form in conjunction with other known anti-inflammatory mediators (GOPO) in this specific sub-species of Rosehip (developed on Langeland, Denmark) has reputed beneficial effects for cartilage and for osteaoarthritis in Man [30, 31] and preliminary data suggests a beneficial effect in working horses .
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